KR101893473B1 - Nd2 peptides and methods of treating neurological disease - Google Patents

Abstract

The present invention is based in part on identifying the core region of ND2 responsible for the interaction with Src within residues 289-321 of ND2, more particularly residues 310-321 of ND2. Peptides that comprise, overlap, or fall within the region can be used to inhibit ND2 interaction with Src. The inhibition of such interactions is useful for the treatment or prevention of neurological diseases and disorders, pain and cancer.

Description

[0001] ND2 PEPTIDES AND METHODS OF TREATING NEUROLOGICAL DISEASE [0002]

Before and after reference to related application - Reference

This application claims the benefit of U.S. Provisional Application No. 61 / 387,439, which is incorporated by reference in its entirety for all purposes.

background

The N-methyl D-aspartate receptor (NMDAR) complex contains more than 60 proteins [4]. NMDAR complexes have been reported to be associated with multiple neurological diseases and disorders, including stroke, neurotrauma, neurodegenerative diseases, memory and long term potentiation, eye and ear neuropathy, and pain. However, efforts to directly inhibit NMDAR complexes have failed clinically due to excessive side effects.

The post-synaptic density protein 95 kD (PSD95) binds directly to the C-terminal NR2 subunit [11] of NMDAR via the first two PDZ domains PDZ1 and PDZ2 [12]. Disruption of the interaction between PSD95 and NMDAR has been reported to protect animals from stroke damage results without interrupting NMDAR's electrical and calcium-flow activity [13].

NADH dehydrogenase subunit 2 (ND2) has been reported to be associated with tyrosine kinase Src (see Figure 1A). Src is one of several Src family kinases (SFK) (ie Src, Fyn, Lyn and Yes) in the NMDAR complex [5-7]. Src is involved in the regulation of many functions, including cell adhesion, growth, migration and differentiation. Src is widely expressed in many cell types and can have a variety of locations within the cell. The subcellular location of Src seems to influence its function. Src can be associated with cell membranes such as plasma membranes, perinuclear membranes, and endosomal membranes. In the plasma membrane, Src can transfer signals from the various receptors to the internal signal transduction pathways, which transmit the signals to the nucleus, cytoskeleton and other cellular components. For example, Src can act through growth factor receptors and affect cell growth and proliferation.

The putative molecular arrangement between NMDAR, Src and ND2 is described in Liu et < RTI ID = 0.0 > al . , Nat Med 2008], where Src is presumed to interact with NMDAR through ND2, which acts as an adapter protein. ND2 modulates NMDA receptor activity by attaching Src to the N-methyl-d-aspartate (NMDA) receptor complex in post-synaptic density (PSD). A fragment of Src, referred to as Src 40-49-Tat, has been reported to inhibit the interaction of Src and ND2 in the brain's excitatory synapse, thereby reducing the phosphorylation of the NR2B subunit and regulating pain [1, 2, 3] .

Outline of claimed invention

The present invention provides an ND peptide having an amino acid sequence consisting of amino acids 310 to 321 of SEQ ID NO: 60, wherein no more than six amino acids of the ND peptide can be deleted, inserted, or conservatively substituted. An ND2 peptide comprising any of the peptides mentioned above may have an amino acid sequence consisting of 4-12 consecutive residues between amino acids 310 to 321 of SEQ ID NO: 60. Optionally, the ND2 peptide has an amino acid sequence consisting of 4-10 consecutive residues between amino acids 310 to 321 of SEQ ID NO: 60. Optionally, the ND2 peptide consists of amino acids 310 to 321. Any of the above ND peptides can be lipidated, for example, by being linked to a fatty acid. Preferably, the ND2 peptide can be myristoylated. Any of the ND2 peptides can be linked to, for example, the N-terminal or C-terminal internalizing peptide of the ND2 peptide as a fusion protein. The internalizing peptide may comprise at least 5 arginines or lysine residues and has a total length of 15 or fewer amino acids. The peptides may be Tat peptides.

The present invention provides chimeric peptides of up to 50 amino acids in length. The peptide comprises an ND2 peptide comprising at least three consecutive amino acids located between amino acids 289 to 321 of SEQ ID NO: 60. The ND2 peptide is linked to the internalizing peptide and / or the ND2 peptide is lipidated. Optionally, the chimeric peptide is less than 25 amino acids in length. Optionally, the ND2 peptide has an amino acid sequence consisting of 4-20 consecutive residues between amino acids 289-321 of SEQ ID NO: 60. Optionally, the ND2 peptide has an amino acid sequence consisting of 4-12 consecutive residues between amino acids 310 to 321 of SEQ ID NO: 60. Optionally, the ND2 peptide has an amino acid sequence consisting of 4-10 consecutive residues between amino acids 310 to 321 of SEQ ID NO: 60. Optionally, the ND2 peptide consists of amino acids 310 to 321 of SEQ ID NO: 60, and up to six amino acids of such ND2 peptide may be deleted, inserted, or substituted. Optionally, the ND2 peptide has an amino acid sequence consisting of amino acids 310 to 321 of SEQ ID NO: 60. Optionally, the internalizing peptide is linked to the N-terminus of the ND2 peptide. Optionally, the internalizing peptide is linked to the C-terminus of the ND2 peptide. Optionally, the internalizing peptide and the ND2 peptide are linked as fusion proteins. Optionally, the internalizing peptide comprises at least 5 arginines or lysine residues and has a total length of no more than 15 amino acids. Optionally, the internalizing peptide is a Tat peptide. The present invention further provides an ND2 peptide having 4-40 residues identical to the residues of SEQ ID NO: 60, wherein at least four of such residues of the ND2 peptide are contiguous between amino acids 289 to 321 of SEQ ID NO: 60 Lt; / RTI >

The present invention further provides a peptidomimetic of the chimeric peptides or ND2 peptides described above. Optionally, the peptidomimetic is a retro-inverso peptidomimetic.

The present invention provides a method of treating or preventing a neurological disease or disorder, comprising administering to a patient having or at risk of developing a neurological disorder a chimeric peptide, an ND2 peptide or an effective remedy of peptidomimetic as described above And the like. Optionally, the neurological disease or disorder is stroke, traumatic injury to the CNS, epilepsy, anxiety, or neurodegenerative disease.

The present invention provides a method of treating or preventing pain, comprising administering to a patient having or at risk of developing pain an effective regimen of the chimeric peptide, ND2 peptide, or peptidomimetic described above Additional information is provided. Optionally, the pain is neuropathy or inflammatory pain.

The present invention provides a method of treating or preventing cancer, comprising administering to a patient having cancer or at risk of developing cancer an effective regimen of the chimeric peptide, ND2 peptide or peptidomimetic described above .

The present invention further provides a method of identifying an agent that inhibits ND2-Src interaction, comprising contacting said agent with a Src peptide and an ND2 peptide, and determining the binding between the Src peptide and the ND2 peptide Wherein the decrease in binding in the presence of the agent relative to the agent-deficient control assay indicates that the agent is an inhibitor of the Src-ND2 interaction, wherein the agent is a chimera or a chimera as defined herein or elsewhere herein ND2 < / RTI > peptide or a peptidomimetic thereof.

The present invention further provides a method for identifying an agent that inhibits Src-ND2 interaction, comprising contacting said agent with said Src peptide and an ND2 peptide as defined above, and binding between the Src peptide and the ND2 peptide Wherein the reduction in binding in the presence of the agent compared to the agent-deficient control assay indicates that the agent is an inhibitor of the Src-ND2 interaction. The method may also include the step of testing the agent for pharmacological activity against a neurological disease, pain or cancer in an animal model of one of neurological disease, pain or cancer.

Brief Description of Drawings

Figures 1A, B and C: ND2 interacts with Src. A. Shown is the interaction in the Src-ND2-NMDAR complex. B. Structure of various ND2 sequences designed to identify Src-interacting domains. C: Dot blot showing that the Src-interacting sequence of ND2 is between amino acids 239-321.

2A, B and C: A. Structure of the ND2 fragment used in the experiment, wherein the numbers indicate amino acids relative to the full-length ND2. B. Dot blots indicating that bioditylated Src 40-58 can bind to ND2, ND2.1 and ND2.1.4 better than ND2.1.3, but not to scrambled sSRC 40-58. C. ELISA indicating that both ND2, ND2.1 and ND2.1.4 can bind to biotinylated Src 40-58.

3A-C: A. Sequence of ND2 constructs determined against binding to Src 40-58. B. Dot blots showing the binding of the ND2 sequence to Src 40-58. C. ELISA showing binding of the ND2 construct to Src 40-58, but not binding to the scrambled control.

4A, B: Dot blots of ND2 fragments showing binding to Src 40-58 with strong binding from ND2 310-321, ND2 307-318, and ND2 310-318. B. ELISA showing binding of ND2 constructs to Src 40-58.

5A-E: A. Dot blot showing that the biotinylated ND2 310-321 is able to bind to the form of Src 40-49 and Src 40-49 with Tat at the amino or carboxy terminus. B. ELISA showing the same as A above, including binding to Src 40-58. C. ELISA assay showing that Src 40-49 can bind Tat-ND2 310-321. D-E. ELISA assays showing that Src 40-49 and Tat Src 40-49 can compete for binding to ND2 310-321 or Tat ND2 310-321.

Figure 6: ELISA assay showing that Src 40-58 can bind Tat-ND2 310-321 more strongly than ND2 310-321.

Figure 7: ELISA showing that Tat-ND2 310-321 can compete for binding to Src 40-49 bound to ND2 310-321.

8A, B: Quantification of co-localization of ND2 and Src in 14DIV hippocampal neurons treated or not treated with Tat-ND2 310-321 or Src 40-49-Tat.

Figures 9A-F: Quantitation of co-localization of proteins in NMDAR complexes in 14DIV hippocampal neurons treated or not treated with Tat-ND2 310-321 or Src 40-49-Tat. A. ND2 and NR2B. B. NR2B and ND2. C. ND2 and PSD95. D. PSD95 and ND2. E. NR2B and PSD95. F. PSD95 and R2B.

Figure 10: Quantification of co-localization of Src and NMDAR 2B in 14 DIV hippocampal neurons treated with Tat-ND2 310-321 or Src 40-49-Tat or not.

Figure 11: Quantification of co-localization of Src and PSD95 in 14DIV hippocampal neurons treated or not treated with Tat-ND2 310-321 or Src 40-49-Tat.

12A-E. Immunoprecipitation experiments from rat brain lysates showing that antibodies against ND2, Src, PSD95, NR2B and NR1 can immunoprecipitate all of the complexes containing other proteins.

13A-D. A. Immunoprecipitation with anti-Nr2B antibodies from 14DIV hippocampal neurons treated with control, Tat-ND2 310-321 or Src 40-49-Tat at 1 μM for 1 hour. B. Repetition of A above using the indicated antibody to A. C. IP treated with 3 [mu] M for 2 hours. D. Using the anti-PSD95 as an immunoprecipitating antibody,

14A, B: ND2 310-321 inhibited PACAP-enhanced NMDA-induced current, while Src 40-49 did not.

Figure 15: Co-immunoprecipitation with anti-Nr2B antibodies from rat brain applied to treatment with 3PVO or 3PVO and 3 [mu] M Tat-ND2 310-321. C - contralateral brain extract; I - ipsilateral brain extract. The label indicates the used detection antibody, and P-Tys indicates the antibody against the phosphorylated tyrosine of Src.

16A, B: Co-immunoprecipitation with anti-PSD95 or anti-NR2B antibodies, indicating the state of the NR2B complex and protein in the presence or absence of Src-Tat or Tat-ND2.

Figure 17: Treatment with Tat-ND2 310-321 reduces pain hypersensitivity in models of CFA-induced pain.

Figure 18: Infarct size of rats applied to 3PVO in the presence of Tat-NR2B9c, src 40-49-Tat, or Tat-ND2 310-321.

Figure 19: Sequence of ND2 with predicted membrane topology. The location of 310-321 is highlighted and is predicted to reside within the cell.

Figure 20: Effect of Tat-ND2, mistroylated ND2 and NA-1 (also known as Tat-NR2B9c) on blood pressure when intravenously injected into rats at high concentrations.

Figure 21: Graph showing the minimum blood pressure (maximum blood pressure drop) observed after intravenous injection of NA-1, Tat-ND2 or myr2-ND2.

Figure 22: Graph showing the effect of Tat-ND2, myr-ND2 and myr2-ND2 on rat brain protection against stroke as induced in the 3PVO model when given intravenously 1 hour after the onset of stroke.

Figure 23: A graph showing that two different concentrations of myr-ND2 can significantly reduce pain by measuring innocuous stimulation pain by paw withdrawal threshold in animals applied to peripheral nerve damage.

Justice

By " chimeric peptide " is meant a peptide having two component peptides that are not naturally associated with each other, either as fusion proteins or linked by chemical bonds.

A " fusion " protein or polypeptide represents a single contiguous amino acid sequence consisting of a complex polypeptide, i.e., a sequence from two (or more) distinct heterologous polypeptides that are not commonly fused together into a single polypeptide sequence.

Agents are usually provided in a separate form. Separated is the need to exclude the presence of other components in order to work with the separated species, such as the internalizing peptide or pharmaceutical excipient, although the target species (e.g., peptide) is naturally associated or used in the production of the target species Means at least partially separated from the contaminant. Preferably, the agonist is a predominant polymeric (e.g., polypeptide) species present in the sample (on a molar basis in the composition), which typically comprises at least about 50 percent (by mole) of all polymeric species present. Generally, the separate pharmacological agent comprises more than 80 to 90 percent of all polymeric species present in the composition. Most preferably, the pharmacological agent is essentially homogeneous, and the composition is essentially composed of a single polymeric species so that the tablet (i. E., The contaminant species can not be detected in the composition by conventional detection methods).

The term " specific binding " refers to a molecule (ligand) that associates with another specific molecule (receptor) in the presence of many different molecules, i.e., exhibits the preferential binding of one molecule to another molecule in a heterogeneous mixture of molecules. E. G., Ligand and receptor, which is characterized by the ability of two molecules, e. The specific binding of the ligand to the receptor is also evidenced by the decreased binding of the detectably labeled ligand to the receptor under the presence of an excess of unlabeled ligand (i. E., Binding competition assay). Specific binding may be the result of the formation of a bond between a particular functional group or a particular spatial fit (e.g., lock and key type), while non-specific binding is usually the result of van der Waals forces.

Excitotoxicity is a pathological process whereby neurons are damaged and killed by overactivation of glutamate receptors, e. G. NMDA receptors.

The term " patient " or " subject " includes humans and other mammals, especially rodents, cats, dogs, ungulates, pigs and non-human primates.

The term " agonist " includes any component, compound, or entity that may or may not have pharmacological activity. Agents may especially be biological agents (e. G., Peptides, peptidomimetics, or antibodies) or organic small molecules (usually less than 500 Da). The agonist may be a product of a natural or synthetic compound. Agents include those that are known (i.e., approved by a similar population in FDA or other countries), compounds that have been identified for pharmacological activity but are being further evaluated, or compounds that are screened for pharmacological activity.

Agents may be described as having pharmacological activity if they exhibit activity in a screening system indicating that the active agent may be useful or useful in the prevention or treatment of disease. The screening system may be an in vitro, cell, animal or human screening system. Agents may be described as having pharmacological activity, although further testing may be required to establish actual prophylactic or therapeutic utility in the treatment of the disease.

Unless otherwise clear from the context, reference to an agonist refers to an agonist or pharmacological agent either alone or linked to an internalizing peptide.

A Tat (or TAT or tat) peptide is a GRKKRRQRRR (SEQ ID NO: 5) in which no more than 5 residues in the sequence have been deleted, substituted or inserted, retaining the ability to promote the uptake of the linked peptide or other agent 60). ≪ / RTI > Preferably, any amino acid change is a conservative substitution. Preferably, any substitution, deletion or internal insertion in the aggregate leaves a peptide with a net cationic charge, preferably similar to the net cationic charge of the sequence. The amino acid of the Tat peptide may be derivatized with biotin or a similar molecule to reduce the inflammatory response.

Statistically significant indicates a p-value of <0.05, preferably <0.01, most preferably <0.001.

When a peptide or amino acid sequence is referred to as occurring within a certain range or within an amino acid, the peptide may include an amino acid in between, as well as a starting point and an ending point that define the range.

For purposes of classifying amino acid substitutions with conservative or non-conservative properties, amino acids can be grouped as follows: Group I (hydrophobic side chain); met, ala, val, leu, with; Group II (neutral hydrophilic side chain): cys, ser, thr; Group III (acid side chain): asp, glu; Group IV (basic side chain): asn, gln, his, lys, arg; Group V (residues affecting chain forms): gly, pro; And Group VI (aromatic side chain): trp, tyr, phe. Conservative substitutions include substitutions between amino acids within the same group. Non-conservative substitutions consist in replacing one member of the group with another member of the group.

The peptides are aligned with the reference sequence maximally when the number of correct matches between the peptide and the reference sequence is maximized. Alignment can be performed with the naked eye. Alternatively, software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). Typically, default program parameters may be used. For the amino acid sequence, the BLASTP program uses a wordlength (W) of 3, an expectation of 10 (E), and a BLOSUM62 scoring matrix by default (Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89, 10915 (1989)).

Peptides occurring within a specific range of amino acids may include peptides comprising only one or both of the amino acids defining the limits of the range, as well as peptides containing only amino acids between the ranges defining the amino acids.

details

I. General

The present invention is based in part on identifying the core region of ND2 responsible for the interaction with Src within residues 289-321 of ND2, more particularly residues 310-321 of ND2. A peptide comprising, or overlapping with, or derived from within said region can be used to inhibit ND2 interaction with Src. Inhibition of such interactions is useful for the treatment or prevention of neurological diseases and disorders, pain and cancer.

II. protein

Unless otherwise clear from the context, the ND2 protein represents the natural human form of ND2, wherein an exemplary sequence is designated in Swiss Prot P03891 and reproduced below and in FIG. The original M residue can be cleaved. Approximately 20 single amino acid natural variants of the sequence are listed in the Swiss-Prot database.

Likewise, unless otherwise specified, Src means the natural human sequence of Src, for example, the natural human sequence of Src with or without the first Met residue is referred to as Swiss-Prot. Lt; / RTI &gt;

III. agent

Agents of the invention may comprise residues 289-321 of the ND2 protein (SEQ ID NO: 60), overlapping residues 289-321, residues 289-321, residues 289-321, preferably residues 289-321, The ND2 peptide comprising residues 310-321 of the ND2 protein, overlapping residues 310-321, consisting of residues 310-321, or residing within residues 310-321. The ND2 peptide usually has at least three consecutive residues within residues 289-321 of ND2. The ND2 peptide preferably contains approximately amino acids 40-49 of Src, or binds to a Src protein within a unique domain at a site within amino acids 40-49 and competitively inhibits interaction with ND2 protein and Src protein. The ND2 peptide typically has 10, 11, 12, 15, 20, 30 or fewer than 40 residues of SEQ ID NO: 60, indicating that when the indicated number of residues in the ND2 peptide is aligned to full length ND2 sequence, &Lt; / RTI &gt; identical to the corresponding residue in the ND2 sequence. Preferably, at least 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 of the residues are continuous within residues 289-321 of ND2, preferably residues 310-321. Preferably, the peptide has 4-20 amino acids identical to the corresponding residue from the ND2 sequence, more preferably 4-12 or 5-10 residues, when maximally aligned with the ND2 sequence. Some ND2 peptides have an amino acid sequence consisting of 4-20 consecutive residues between amino acids 289-321 of SEQ ID NO: 60. Some ND2 peptides have an amino acid sequence consisting of 3-12 consecutive residues between amino acids 310 to 321 of SEQ ID NO: 60. Some ND2 peptides have an amino acid sequence consisting of at least 3, 4, 5, 6, 7, 8, 9, 10, 11 residues having amino acids 310-321 of SEQ ID NO: Some ND2 peptides consist of residues 310-321 of SEQ ID NO: 60.

To facilitate membrane traversal and serve as a tag such as biotin or GST and to aid in purification, identification or screening, the flaking amino acids not associated with SEQ ID NO: 60, as discussed below, For example, an internalizing peptide. Except for the unrelated flanking sequence of amino acids, any amino acid in the ND2 peptide that differs from SEQ ID NO: 60 is preferably a conservative substitution with respect to the corresponding residue of SEQ ID NO: 60. An ND2 peptide (not including an unrelated flanking sequence) having a sequence that differs from SEQ ID NO: 60 preferably has 6, 5, 4, 3, 2 or 1 or fewer Deletion, insertion or substitution. The ND2 peptide preferably has no more than 40, 30, 20, 15, or 12 amino acids in total (not involving flanking sequences, e.g., an internalizing peptide).

Agents of the invention also include peptidomimetics of ND2 peptides. The peptidomimetic is a synthetic chemical compound having substantially the same structural and / or functional characteristics as the ND2 peptide consisting of natural amino acids but having at least one non-peptide bond or at least one non-natural amino acid.

The peptidomimetics may contain the entire synthetic non-natural analog of the amino acid, or may be a chimeric molecule of the natural peptide amino acid and, in part, the non-natural analog of the amino acid. The peptidomimetics may also contain any amount of natural amino acid conservative substitutions so long as the substitutions also do not substantially alter the mimetic structure and / or inhibitory or binding activity. In peptidomimetics of chimeric peptides comprising ND2 peptides and internalizing peptides, the active moieties or internalizing moieties, or both, may be peptidomimetic.

The peptides and peptidomimetics of the present invention may contain modified amino acid residues, for example, N-alkylated residues. N-terminal alkyl modifications include for example N-methyl, N-ethyl, N-propyl, N-butyl, N-cyclohexylmethyl, N-cyclohexylethyl, Phenylpropyl, N- (3,4-dichlorophenyl) propyl, N- (3,4-difluorophenyl) propyl, and N- (naphthalen-2-yl) ethyl). The peptide or peptidomimetic can also be acetylated and / or phosphorylated and / or glycosylated.

In some peptidomimetics, any amino acid naturally occurring in the L-form (which may also be referred to as R or S, depending on the structure of the compound) may be replaced by an amino acid of the same chemical structural type or peptidomimetic, May be replaced by an amino acid of the opposite chirality commonly referred to as D-amino acid, which may be further referred to as form or S-form. Thus, the peptidomimetic may comprise 1, 2, 3, 4, 5, at least 50%, or all D-amino acid residues. Peptidomimetics containing some or all of the D residue are often referred to as " inco " peptides.

Peptidomimetics also include retro peptides. Retropeptides have reverse amino acid sequences. Peptidomimetics also include retroincomplexes in which the sequence of amino acids is reversed as the native C-terminal amino acid is seen at the N-terminus and the D-amino acid is used instead of the L-amino acid.

The individual peptidomimetic moieties may be linked by peptide bonds, other chemical bonds or coupling means such as glutaraldehyde, N-hydroxysuccinimide ester, biologically active maleimide, N, N-dicyclohexylcarbodiimide ( DCC) or N, N-diisopropylcarbodiimide (DIC). The linking group, which may be an alternative to conventional amide linkage (" peptide bond ") linkages, is, for example, -C (= O) -CH2- ), Aminomethylene (CH2-NH), ethylene, olefin (CH = CH), ether (CH2-O), thioether (CH2-S), tetrazole (CN4-), thiazole, retroamide, Or esters (see, for example, Spatola (1983) in Chemistry and Biochemistry of Amino Acids, Peptides and Proteins, Vol.7, pp 267-357, A Peptide Backbone Modifications, Marcell Dekker, NY).

Mimetics of aromatic amino acids include, for example, D- or L-naphylalanine; D- or L-phenylglycine; D- or L-2 thienyl alanine; D- or L-1, -2, 3-, or 4-pyrenylalanine; D- or L-3 thienylalanine; D- or L- (2-pyridinyl) -alanine; D- or L- (3-pyridinyl) -alanine; D- or L- (2-pyrazinyl) -alanine; D- or L- (4-isopropyl) -phenylglycine; D- (trifluoromethyl) -phenylglycine; D- (trifluoromethyl) -phenylalanine; D-p-fluorophenylalanine; D- or L-p-biphenyl phenylalanine; K- or L-p-methoxybiphenylphenylalanine; D- or L-2-indole (alkyl) alanine; And D- or L-alkylalanine wherein the alkyl is substituted or unsubstituted methyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl, iso-butyl, sec-iso-butyl, - &lt; / RTI &gt; acidic amino acid). The aromatic rings of the unnatural amino acids include, for example, thiazolyl, thiophenyl, pyrazolyl, benzamidazolyl, naphthyl, furanyl, pyrrolyl, and pyridyl aromatic rings.

Mimetics of acidic amino acids include, for example, non-carboxylate amino acids while retaining negative charge; (Phosphono) alanine; Can be produced by substitution with sulfated threonine. Carboxyl side groups (e.g., aspartyl or glutamyl) also include carbodiimide (RNCNR =), such as 1-cyclohexyl-3 (2-morpholinyl- Amide or 1-ethyl-3 (4-azonia-4,4-dimetolpentyl) carbodiimide. Aspartyl or glutamyl can also be converted to Lt; RTI ID = 0.0 &gt; glutaminyl &lt; / RTI &gt; residues.

The mimetics of basic amino acids are, for example, the amino acid ornithine, citrulline or (guanidino) -acetic acid, or (guanidino) alkyl-acetic acid (in addition to lysine and arginine) And the like). Nitrile derivatives (e.g., containing a CN-moiety in place of COOH) can be substituted for asparagine or glutamine. The asparaginyl and glutaminyl residues may be deaminated with the corresponding aspartyl or glutamyl residues.

The arginine residue mimetic is preferably selected from the group consisting of arginyl and one or more conventional reagents including alkaline, such as phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, or ninhydrin, &Lt; / RTI &gt;

Tyrosine residue mimetic can be produced by reacting tyrosyl, for example, an aromatic diazonium compound or tetranitromethane. N-acetylimidizole and tetranitromethane can be used to form O-acetyl tyrosyl species and 3-nitro derivatives, respectively.

The cysteine residue mimetic is reacted with a cysteinyl residue, for example, alpha-haloacetate, e.g., 2-chloroacetic acid or chloroacetamide, and the corresponding amine to produce a carboxymethyl or carboxyamidomethyl derivative . &Lt; / RTI &gt; The cysteine residue mimetic can also be prepared by reacting a cysteinyl residue with, for example, bromo-trifluoroacetone, alpha-bromo-beta - (5-imidazoyl) propionic acid; Chloroacetyl phosphate, N-alkylmaleimide, 3-nitro-2-pyridyl disulfide; Methyl 2-pyridyl disulfide; p-chloromercibenzoate; 2-Chloromercury-4-nitrophenol; Or chloro-7-nitrobenzo-oxa-1,3-diazole.

Lysine mimetics can be produced by reacting lysinyl with, for example, succinic anhydride or other carboxylic acid anhydrides (and amino terminal residues can be changed). Lysine and other alpha-amino-containing residue mimetics may also be used in conjunction with imidoesters such as methylpicolinimidate, pyridoxalphosphate, pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid, O-methylisourea, 2,4, pentanedione, and transamidase-catalyzed reaction with glyoxylate.

The mimetic of methionine can be produced, for example, by reaction with methionine sulfoxide. The mimetics of proline can be, for example, selected from the group consisting of piperolic acid, thiazolidinecarboxylic acid, 3- or 4-hydroxyproline, dehydrofroline, 3- or 4-methylproline, . The histidine residue mimetic may be produced by the reaction of histidyl with, for example, diethyl carbonate or para-bromophenacyl bromide.

Other mimetics include, for example, hydroxylation of proline and lysine; Phosphorylation of the hydroxyl group of a seryl or threonyl residue; Methylation of alpha-amino groups of lysine, arginine and histidine; Acetylation of the N-terminal amine; Methylation of the backbone amide residue or substitution with N-methyl amino acid; Or amidation of a C-terminal carboxyl group.

Linkers, e. G., Polyethylene glycol linkers, can be used to duplex ND2 peptides or peptidomimetics to enhance affinity and selectivity for Src. Optionally, about 2-10 copies of PEG can be serially serially linked as linkers.

The appropriate pharmacological activity of the peptides, peptidomimetics or other agonists can be determined, if desired, by demonstrating inhibition of the Src-ND2 interaction in vitro or in animal models as described below. Useful peptides or peptidomimetics typically have an IC50 value of less than 50 [mu] M, 25 [mu] M, 10 [mu] M, 0.1 [mu] M or 0.01 [mu] M in the assay. Preferred peptides typically have an IC50 value of 0.001 to 1 [mu] M, more preferably 0.05 to 0.5 or 0.05 to 0.1 [mu] M.

IV. Internalizing peptides and lipidation

Internalization peptides, also known as cell membrane transduction peptides or cell penetration peptides, are relatively short (e.g., 5-30 or 7-20 or 9-15 amino acids) peptides that allow many cellular or viral proteins to cross the membrane &Lt; / RTI &gt; Such peptides typically have a cationic charge from the standard representations of arginine and / or lysine residues (generally associated with proteins) that are believed to promote the passage across the membrane. Some such peptides have at least 5, 6, 7 or 8 arginine and / or lysine residues. Examples include the antennapedia protein (Bonfanti, Cancer Res. 57, 1442-6 (1997)) (and variants thereof), the Tat protein of human immunodeficiency virus, the protein VP22, the product of the UL49 gene of herpes simplex virus type 1 , Penetratin, SynBl and 3, Transportan, Amphipathic, gp41NLS, polyArg, and various plant and bacterial protein toxins, such as ricin, abrin ), Modeccin, diphtheria toxin, cholera toxin, anthrax toxin, heat labile toxin, and Pseudomonas aeruginosa exotoxin A (ETA). Other examples are described in the following references (Temsamani, Drug Discovery Today, 9 (23): 1012-1019, 2004; De Coupade, Biochem J., 390: 407-418, 2005; Saalik Bioconjugate Chem. : 1246-1253, 2004; Zhao, Medicinal Research Reviews 24 (1): 1-12, 2004; Deshayes, Cellular and Molecular Life Sciences 62: 1839-49, 2005).

A preferred internalizing peptide is Tat from HIV virus. Tat peptides reported in previous work include or consist of the standard amino acid sequence YGRKKRRQRRR (SEQ ID NO: 2) found in HIV Tat protein. SEQ ID NO: 2 is designated as the standard Tat peptide. If additional moieties flanked by the Tat motif are present (in addition to a pharmacological agent), the moiety may be, for example, a Tat protein of the type commonly used to link two peptide domains, spacer or linker amino acids (SEQ ID NO: 44), TGEKP (SEQ ID NO: 45), GGRRGGGS (SEQ ID NO: 46), or LRQRDGERP (SEQ ID NO: : 47) (for example, Tang et al . (1996), J. Biol. Chem. 271,15682-15686; Hennecke et al . (1998), Protein Eng. 11, 405-410) or may be any other amino acid that does not significantly diminish the ability to provide absorption of variants without flanking residues. Preferably, the number of flanking amino acids that are not active peptides does not exceed ten in any aspect of YGRKKRRQRRR (SEQ ID NO: 2). A suitable Tat peptide is YGRKKRRQRRRPQ (SEQ ID NO: 48), as long as it contains additional amino acid residues flanked at the C-terminus of YGRKKRRQRRR (SEQ ID NO: 2). Preferably, however, no flanking amino acid is present.

Variants of the Tat peptide with reduced ability to bind to N-type calcium channels are described in WO / 2008/109010. Such variants may comprise or consist of the amino acid sequence XGRKKRRQRRR (SEQ ID NO: 49), wherein X is an amino acid other than Y or is absent (in which case G is a free N-terminal residue). Preferred Tat peptides have an N-terminal Y residue substituted with F. Thus, a tat peptide comprising or consisting of FGRKKRRQRRR (SEQ ID NO: 3) is preferred. Another preferred mutant tat peptide is composed of GRKKRRQRRR (SEQ ID NO: 1). Other tat peptides that may be used include GRKKRRQRRRPQ (SEQ ID NO: 4) and GRKKRRQRRRP (SEQ ID NO: 59). Other Tat peptides include at least eight consecutive amino acids of the sequence GRKKRRQRRR. Other tat peptides which do not inhibit the N-type calcium channel and promote absorption of the agonist include those mentioned above. Another preferred Tat peptide is referred to as rv-Tat or RRRQRRKKRGY (SEQ ID NO: 58).

X may represent a free amino terminus, one or more amino acids, or a conjugated moiety. Internalized peptides can be used in the form of Inverso or Retro or Inverso retro, wherein the linked peptide or peptidomimetic is present or absent.

The internalized peptide may be attached to the agent by conventional methods. For example, an agonist may be linked to the internalizing peptide by chemical bonding, for example, via a coupling or conjugation agent. A number of such agents are commercially available and are outlined in Wong, Chemistry of Protein Conjugation and Cross-Linking, CRC Press (1991). Some examples of crosslinking reagents are J-succinimidyl 3- (2-pyridyldithio) propionate (SPDP) or N, N '- (1,3-phenylene) bismaleimide; N, N'-ethylene-bis- (iodoacetamide) or other such reagents having 6 to 11 carbon-methylene bridges (relatively specific to sulfhydryl groups); And 1,5-difluoro-2,4-dinitrobenzene (which forms an irreversible bond with the amino and tyrosine groups). Other crosslinking reagents include p, p'-difluoro-m, m'-dinitrodiphenyl sulfone (which forms an irreversible crosslink with the amino and phenolic groups); Dimethyl adipimidate (specific for amino group); Phenol-1,4-disulfonyl chloride (mainly reacts with an amino group); Hexamethylene diisocyanate or diisothiocyanate, or azophenyl-p-diisocyanate (mainly reacting with an amino group); Glutaraldehyde (which reacts with several different side chains) and distyazo benzidine (which reacts mainly with tyrosine and histidine).

Peptide attachment of an agonist to an internalizing peptide can be accomplished by generating a fusion protein, preferably comprising a peptide sequence fused to the internalizing peptide at the N-terminus.

Peptides optionally fused to a Tat peptide can be synthesized by solid phase synthesis or recombinant methods. Peptidomimetics are described in the scientific and patent literature, for example Organic Syntheses Collective Volumes, Gilman et &lt; RTI ID = 0.0 &gt; al . (Eds.) John Wiley &amp; Sons, Inc., NY, al-Obeidi (1998) Mol . Biotechnol . 9: 205-223; Hruby (1997) Curr . Opin . Chem. Biol 1: 114-119; Ostergaard (1997) Mol . Divers . 3: 17-27; Ostresh (1996) Methods Enzymol . 267: 220-234. &Lt; / RTI &gt; Peptides or peptidomimetics as fusion peptides or otherwise linked to internalizing peptides preferably comprise no more than 50 total amino acids, more preferably no more than 25 or no more than 20 amino acids.

In addition to linking the ND2 peptide to the internalizing peptide or in addition to linking the ND2 peptide to the internalizing peptide, the ND2 peptide can be linked (lipidated) to the lipid to cross the cell membrane by increasing the hydrophobicity of the conjugate relative to the peptide alone, Crossing or crossing the brain barrier. &Lt; / RTI &gt; Lipidation is preferably carried out on the N-terminal or C-terminal amino acid, but may also be performed on the internal amino acid unless the binding constant of the ND2 peptide to Src is reduced by more than 50%. Lipids are organic molecules that are more soluble in the ether than water, including fatty acids, glycerides and sterols. Suitable forms of lipidation include, but are not limited to, the attachment of other fatty acids having a chain length of 10-20 carbons such as myristoylation, palmitoylation or preferably lauric acid and stearic acid, as well as geranylation, &Lt; / RTI &gt; geranylgeranylation, and isoprenylation. The type of lipidation that occurs in post-translational modifications of natural proteins is desirable. Lipidation with fatty acids through formation of an amide bond to the alpha-amino group of the N-terminal amino acid of the peptide is also preferred. Lipidation can be carried out by peptide synthesis involving the pre-lipidated amino acid, by in vitro enzymatic or recombinant expression, or by chemical cross-linking or chemical derivatization of the peptide . Amino acids modified by myristoylation and other lipid modifications are commercially available.

Lipidation preferably does not cause a temporary decrease in blood pressure, as found in the case where a standard Tat peptide is administered at high doses (e.g., 3 mg / kg or more), or at least does not lead to standard Tat peptides Facilitates the passage of bound ND2 peptides across the cell membrane and / or the blood brain barrier with less reduction than the same ND2 peptide.

When a temporary decrease in blood pressure occurs in the case of ND2 peptide administration (for example, when it is linked to a standard Tat peptide and administered at a high dose), it may be combined with an anti-inflammatory agent, preferably a co-administration of a mast cell degranulation inhibitor (See for example WO / 2009/07610).

V. Patients who can be treated or preventable

The agents of the present invention are useful for treating or preventing neurological diseases or disorders, pain and cancer. This class is of course not mutually exclusive. For example, brain cancer can belong to all three classes.

Various neurological diseases and disorders can be treated or prevented. Such diseases and disorders include, but are not limited to, anxiety, epilepsy, eye or retinal neuropathy, traumatic injury to the CNS that is not associated with stroke (e.g. spontaneous, acute ischemia, hemorrhagic, procedural stroke), epilepsy, hypoxia, For example, neurotrauma, traumatic brain injury and spinal cord injury, Alzheimer's disease, Parkinson's disease, other dementias associated with Lewy bodies, Huntington's disease, ALS, bovine encephalopathy, Creutzfeldt-Jakob disease, multiple sclerosis, Cerebellar ataxia, Tai-sachs disease, and transmissible spongiform encephalopathy. These disorders may also be associated with patients undergoing surgery that can affect or affect blood vessels (e. G., The neck vein or the carotid artery) that supply blood to the brain or remove blood from the brain, , Intravascular surgery to repair the aneurysm, or patients undergoing intravascular surgery on blood vessels supplying blood to the limbs, spinal cord, retina or kidney. Such restoration can be accomplished, for example, by inserting a stent or coil into a blood vessel that is applied to an aneurysm nervous system disease and disorder at least partially associated with excitotoxicity, which is particularly applicable to treatment by the methods of the present invention.

Stroke is a disease caused by damaged blood flow in the CNS, regardless of the cause. Potential causes include embolism, bleeding, thrombosis, and surgery. Some neurons die immediately as a result of damaged blood flow. These cells release component molecules, including glutamate, which in turn activate NMDA receptors, which increase intracellular calcium levels and intracellular enzyme levels to cause additional neuronal cell death (excitotoxicity cascade). The death of tissue from the deficiency of oxygen is referred to as infarction. The infarct volume (ie, the volume of killed neurons resulting from a stroke in the brain) can be used as an indicator of the degree of pathological damage resulting from a stroke. The symptom outcome depends on the volume of the infarct and the location in the brain where the infarction is located. The Disability Index can be used as a measure of symptom impairment, for example the Rankin Stroke Outcome Scale (Rankin, Scott Med J; 2: 200-15 (1957)), the NTH Stroke Scale, and the Barthel Index. The Rankin scale is based on a direct assessment of the patient's overall condition as follows.

0 No signs at all

1 Significant impairment despite signs; Can perform all unfamiliar tasks and activities

2 slight disability; Can not perform all existing activities, but can supervise their own work without assistance

3 Intermediate disability requiring some assistance, but can be walked without assistance

4 Moderate to severe disability; Unable to walk without assistance, unable to perform their physical demands without assistance

5 Severe disability; Lying, incontinence, and constant nursing and attention needed

The Barthel index is based on a series of questions about the ability of the patient to perform 10 basic activities of daily survival to generate a score of 0-100 and the lower score indicates the addition of disability (Mahoney et al . , Maryland State Medical Journal 14: 56-61 (1965)).

Alternatively, stroke severity / outcome can be measured using the NIH Stroke Scale available on the World Wide Web at ninds.nih.gov/doctors/NIH_Stroke_Scale_Booklet.pdf.

The scale is based on the ability of the patient to perform eleven groups of functions including an assessment of the patient's level of consciousness, motor, sensory and speech function.

An ischemic stroke more particularly represents a type of stroke caused by blockage of blood flow to the brain. The underlying disease for this type of block is most commonly the occurrence of fat deposition inside the vessel wall. Such diseases are referred to as atherosclerosis. Such lipid deposition can cause both types of occlusion. Cerebral hemorrhage represents a blood clot (blood clot) that occurs in a clogged portion of a blood vessel. &Quot; Cerebral embolism " generally refers to a blood clot that forms in another location within the circulatory system, usually in the large arteries of the heart and upper chest and neck. Thereafter, a part of the blood clot is destroyed and released, enters the bloodstream, and travels through the blood vessels of the brain until it reaches a blood vessel too small to pass through. The second most important cause of embolism is irregular heartbeats known as arterial fibrillation. This causes blood clots to form in the heart, move from them, and cause diseases that can migrate to the brain. Additional potential causes of ischemic stroke include, but are not limited to, bleeding, thrombosis, incision of an artery or vein, cardiac arrest, shock of any cause, including bleeding, Or direct surgical injury to the blood vessels. Ischemic stroke accounts for about 83 percent of all stroke patients.

Transient ischemic attack (TIA) is a small stroke or borderline stroke. In TIA, there is a disease that ischemic stroke, and a typical stroke boundary signal is generated. However, clogging (blood clots) occurs for a short period of time and tends to dissolve itself through common mechanisms. Patients undergoing cardiac, pulmonary or neurosurgical procedures are at particular risk of transient ischemic attacks.

Hemorrhagic stroke accounts for about 17 percent of stroke patients. This occurs from weakened blood vessels, which rupture and cause bleeding to the surrounding brain. Blood accumulates and presses the surrounding brain tissue. Two common types of hemorrhagic stroke are intracerebral hemorrhage and subarachnoid hemorrhage. Hemorrhagic stroke results from rupture of weakened blood vessels. Potential causes of rupture from weakened blood vessels include hypertensive hemorrhages where high blood pressure causes rupture of the blood vessels, or another ruptured blood vessel such as a ruptured cerebrovascular anomaly including arteriovenous fistula, AVM or spongiform malformations Include the root cause. Hemorrhagic stroke may also result from hemorrhagic conversion of an ischemic stroke which weakens the blood vessels in the infarct, or bleeding from primary or metastatic tumors in the CNS, which ideally contains weak blood vessels. Hemorrhagic stroke can also result from a causal agent such as direct surgical injury to the blood vessels. Aneurysms are ballooning of weakened areas of blood vessels. If left untreated, the aneurysm will continue to weaken and bleed into the brain until it ruptures. Arteriovenous malformation (AVM) is a group of abnormally formed blood vessels. Cavernous malformation is a vein abnormality that can cause bleeding from a weakened vein structure. Any one of the blood vessels may rupture, and may also cause bleeding to the brain. Hemorrhagic stroke can also result from physical trauma. A hemorrhagic stroke in one part of the brain can cause an ischemic stroke in another part through a lack of blood that is lost in a hemorrhagic stroke.

The agents of the present invention are also useful for the treatment or prevention of pain. Pain is a subjective empirical phenomenon in an individual experiencing it, which is influenced by the mental state of the individual, including the environmental and cultural background. &Quot; Physical " pain can often be associated with perceptible stimuli by third parties that cause actual or potential tissue damage. In this sense, pain may be viewed as "a sensory and emotional experience associated with, or described in connection with, actual or potential tissue damage," according to the International Association for the Study of Pain (IASP) . However, some examples of pain do not have a perceptible cause. For example, mental pain includes occasional persistent perceptual pain syndromes in people with psychological disturbances with no evidence of pre-existing physical pain due to psychogenic factors, or perceptual causes of pain do.

Pain is generally divided into three major classes: physiological, inflammatory, and neuropathic pain. However, a number of mechanisms together with some overlap cause each of these classes because each of the classes applies to neural plasticity or to the expression of neuronal plasticity. Neuronal plasticity is generally divided into activation, regulation, and modification, each of which can cause sensitization to pain stimuli by altering the threshold of sensitivity. Pain is not a passive consequence of the prescribed peripheral input to the pain center in the cortex, but rather an active process that occurs in part in the peripheral nervous system and partly in the central nervous system, due to changes in plasticity.

Physiological pain is initiated by a reaction to a noxious stimulus (needle puncture, extreme temperature, compound), inflammatory pain is initiated by tissue damage / inflammation, and neuropathic pain is initiated by nervous system lesions. Each of which is characterized by an impairment at the site of injury and adjacent normal tissue. In this case, a stimulus that does not normally produce pain produces pain (harmless stimulus pain), and a harmful stimulus (sharp object, heat, compound) produces a larger, more sustained pain (hyperalgesia). Inflammatory and physiologic pain Hypersensitivity generally returns to normal after disease course or lesion return to normal. Neuropathic pain will persist even after the initiation event has healed, which is the result of abnormal neural function, not response to the lesion.

Pain can also be referred to as acute or chronic. Acute pain is a naturally temporary, sharp pain, such as caused by pin pricking. Chronic pain is usually pain or pain sensitivity that lasts for longer periods of time per day or more. A rodent model of chronic pain may include a formalin footpad injection, a complete Freund's adjuvant footpad injection, a neurocompaction model (spine / sciatica), and all neuropathic pain models.

Pain may be caused by a variety of factors, including, but not limited to, invasive pain (including body and visceral pain), neuropathic / neuropathic pain (particularly degenerative, pressure induced, inflammatory, infectious-induced neuropathic / neuropathic pain), sympathetic pain inflammatory pain, Pain, and / or idiopathic pain, neuropathic pain, neuralgia, neuritis, malignant pain, angina pectoris, and / or idiopathic pain , And Complex Regional Pain Syndrome I, II, Complex Regional Pain Syndrome II. These terms are defined by the International Association for the Study of Pain and are not mutually exclusive.

Invasive pain is initiated by specialized sensory impairment receptors in peripheral nerves in response to harmful stimuli, harmful coding stimuli, before action. In general, the nociceptive receptors on A-delta and C fibers are free nerve endings ending in the tendons, joints, and internal organs beneath the skin. The DRG neurons provide communication between the peripheral nerves and the spinal cord. The signal is processed through the spinal cord into the brainstem and thalamus, and finally into the cerebral cortex, where the signal usually (but not always) causes a sense of pain. Inflammatory pain generally refers to a wide variety of chemical, thermal, biological (e.g., inflammatory) or inflammatory conditions that have the potential to irritate or damage body tissues, beyond a certain minimum threshold of intensity needed to cause an invasive activity in an immune receptor Can arise from mechanical events.

Inflammatory pain represents pain associated with inflammation. Inflammation is an organism's immune response to infection, irritation and / or damage. Inflammation is characterized by fever, swelling, and warmth. Pain-induced stimuli often produce an inflammatory response, which can itself be the cause of pain experience.

Neuropathic pain is a result of abnormal function in the peripheral nervous system or central nervous system, which generally causes peripheral or central neuropathic pain, respectively. Neuropathic pain is a pain initiated or caused by a primary lesion or dysfunction in the nervous system, defined by the International Association for Pain Research. Neuropathic pain often involves actual impairment of the nervous system, particularly in chronic cases. Inflammatory infiltrative pain is generally the result of tissue damage and the resulting inflammatory process. Neuropathic pain can persist after apparent healing (e. G., Months or years) of any observable damage to the tissue.

In neuropathic pain, sensory processing from the diseased area can be abnormal, and harmless stimuli (e.g., heat, touch / pressure) that do not generally cause pain can cause pain Pain), harmful stimuli can lead to exaggerated perception of pain (ie, hyperalgesia) in response to common painful stimuli. In addition, electrical impulses or shocks or sensations having a sensation similar to " pins and needles " (i.e., sensory anomalies) and / or unpleasant properties (i.e., anomalous sensations) may be caused by general stimulation. Sudden pain is an exacerbation of pre-existing chronic pain. Hyperalgesia is a painful syndrome resulting from an abnormally painful response to stimuli. In most patients, stimuli are repetitive with increased pain thresholds, which can be regarded as the least pain experience the patient can perceive as pain.

Examples of neuropathic pain include, but are not limited to, tactile harmless pain (e.g., induced after nerve damage) neuralgia (e.g., post herpetic neuropathic pain, trigeminal neuralgia), reflex sympathetic dystrophy / Trauma), factors of cancer pain (e.g., pain due to a related disease such as cancer itself or inflammation, or pain due to treatment such as chemotherapy, surgery or radiation therapy), vesicles, Wrist ulceration syndrome), and neuropathy, e.g., peripheral neuropathy (e.g., diabetes, HIV, chronic alcohol use, exposure to other toxins (including many chemotherapeutic agents), vitamin deficiency, Due to other medical conditions). Neuropathic pain includes pain induced by the manifestation of pathological effects of the nervous system after nerve injury due to various causes, such as surgical operations, wounds, herpes zoster, diabetic neuropathy, cutting of the arm or leg, cancer, do. Medical disorders associated with neuropathic pain include traumatic nerve damage, stroke, multiple sclerosis, spinal cord convulsion, spinal cord injury, and cancer.

In some diseases, pain appears to be caused by a complex mixture of invasive and neuropathic factors. For example, chronic pain often involves inflammatory or neuropathic pain, or a combination of the two. Primary neurological dysfunction or impairment can trigger nerve release of inflammatory mediators and subsequent neuropathic inflammation. For example, migraine headache can be a mixture of neuropathy and invasive pain. In addition, myofascial pain is secondary to invasive input from muscle, but abnormal muscle activity may be the result of neuropathic disease.

Symptoms of pain experienced by a patient may or may not involve symptoms of pain that may be identified by the clinician. Conversely, pain may manifest as clinical symptoms in which the patient does not recognize the signs. Symptoms of pain may include, for example, a response to a pain of behavior change type. Exemplary responses to pain include conscious avoidance of painful stimuli, protective responses to protect the body or parts of the body from painful stimuli, reactions to minimize pain, promote healing, minimize pain transmission, and physiological responses . &Lt; / RTI &gt; The delivery response may include vocalization of pain or facial expression or a change in attitude. Physiological responses include reactions that are mediated by the autonomic or endocrine system, for example, enhanced release of adrenaline and noradrenaline, increased production of glucagon and / or hormones and / or corticosteroids. Physiological changes that can be monitored include movement effects such as single contractions, convulsions, paralysis, extended pupil, tremor, sensory irritation and / or altered reflexes. Physiological cardiovascular responses to pain may include changes in blood pressure, changes in pulse rate and characteristics, decreased peripheral circulation, cyanosis, and congestion. Increased muscle tone (tense) is also a sign of pain. Changes in pain-responsive brain function can be monitored by a variety of techniques such as EEG, FEMG, or positron emission tomography (PET).

Another indication of pain may be associated pain, which is the perception of localized pain adjacent or distant to the actual site of the pain-triggering stimulus. Often, associated pain occurs when the nerve is compressed or damaged near its origin or origin. In this situation, the sensation of pain will generally be felt in the area where neurons act, even if damage occurs elsewhere. A common example occurs in the prolapse of the original disc, where the nerve roots originating from the spinal cord are compressed by adjacent disc materials. Pain may occur from the damaged disc itself, but pain may also be felt in areas that act on the stressed nerves (eg, thighs, knees, or feet).

Invasive activity is a sign of invasive pain. Invasive activity can also trigger a variety of autonomic responses, such as avoidance reflexes and pale, sweating, bradycardia, hypotension, lightheadedness, zone and syncope, even in the absence of consciously perceived pain.

The agents of the present invention are also useful for treating cancer or achieving prevention. Src is a tumor gene present in the human body, and many cancers are associated with overexpression, mutation or activity thereof. They particularly include solid tumors, such as breast, colon, lung, prostate, pancreatic, head and neck cancer in particular. Src can also become abnormally active due to mutations in other proteins that regulate it. The methods of the present invention are particularly useful for elevated cancers compared to non-cancerous tissues that are associated with elevated expression of Src at the mRNA or protein level, particularly Src expression, tissue-matched in the same patient. In some methods, the expression of Src in cancer is optionally identified by comparison with the expression of tissue-matched non-cancerous samples from the same patient. However, it is not necessary to confirm the expression level. The detectable activity of Src kinase and elevated activity, in particular compared to histochemically matched non-cancer control samples, provide an indication that the cancer is applicable to treatment using the methods of the present invention. The increased copy number of the Src gene in cancer cells can provide an alternative or additional indicator that cancer is applicable to the treatment. Increased copy numbers can be detected using, for example, Southern blotting, quantitative PCR, fluorescence in situ hybridization, fluorescence of mid-term chromosomal spread, and other cytogenetic techniques. The presence of a mutation in Src associated with carcinogenic activity is also an indicator that cancer can be treated by the method of the present invention.

IX . Treatment / prevention methods

a) Treatment method

Agonists that are optionally attached to an internalizing peptide are administered to a patient with symptom (s) and / or symptom (s) of the disease or disorder described above as therapeutically effective therapy. Such therapy may include administration of at least one of the disease (s) in a patient (or animal model) population suffering from the disease or disorder being treated as compared to a control population of a patient (or animal model) suffering from a disease or disorder not treated with an agent of the invention Dose, route and route of administration which are effective in treating, reducing, or inhibiting the further deterioration of the symptoms or symptoms. The therapy is also considered therapeutically effective when the patient being treated individually achieves a favorable outcome over the average outcome in a control population of equivalent patients not being treated by the methods of the present invention. The number of doses depends on the disease or disorder being treated. Single doses are often sufficient for at least a seizure of the disease, for acute or seizure disorders, such as stroke, traumatic injury, anxiety, acute pain, or epilepsy. For chronic diseases, such as neurodegenerative diseases such as Alzheimer's disease or Parkinson's disease, cancer, or chronic pain, multiple administrations and sometimes lifelong treatment are designated.

For patients suffering from strokes or other ischemic diseases of the CNS, the agent is administered by a regimen including dosages, frequency and route of administration effective to reduce the damaging effects of stroke or other ischemic diseases. If the disease requiring treatment is a stroke, the outcome may be determined by the infarct volume or disability index, and the dose may be determined by the individual patient being treated on the Rankin scale at 2 or less and the Barthel scale at 75 or greater Or a group of patients being treated is considered to be therapeutically effective when they exhibit a significantly improved (i.e., less disordered) distribution of scores on the disorder scale than an equivalent untreated population. See, for example, Lees et al . , &Lt; / RTI &gt; N Engl J Med 2006; 354: 588-600). A single administration of an agent is often sufficient for the treatment of stroke.

The agents of the present invention are also useful for extending the efficacy or safety window of reperfusion, or improving the safety or efficacy of reperfusion at the time provided. This is particularly useful in the treatment of ischemic stroke with another agent that disrupts clots such as tissue plasminogen activator, wherein the window useful for administration is a post-stroke after an increase in the frequency of bleeding events over time It is only 0-4.5 hours. The agents of the present invention may be administered to improve the safety and / or efficacy of agents that disrupt clots in the brain.

Usually, 1 to 8 doses of agonist are administered to treat cancer, but more dose can be provided. Agents may be administered daily, biweekly, weekly, biweekly, monthly or at some other interval, depending on the half-life of the agonist, for example, 1 week, 2 weeks, 4 weeks, 8 weeks, 3-6 months, &Lt; / RTI &gt; Repeated treatments such as chronic administration are also possible.

The treatment of cancer using an agent may be carried out using a conventional therapeutic agent such as Taxol (paclitaxel) or a derivative thereof, a platinum compound such as carboplatin or cisplatin, For example, an anthrocycline such as doxorubicin, an alkylating agent such as cyclophosphamide, an anti-metabolite such as 5-fluorouracil, Can be combined with etoposide. Agents of the invention may be administered in combination with, for example, two, three or more of the above agents in breast cancer and ovarian cancer in standard chemotherapeutic regimes, such as taxol and carboplatin . Other agents for combination include, but are not limited to, biological agents such as Herceptin (TM) for HER2 antigen, Avastin (TM) for VEGF, or monoclonal antibodies including antibodies against EGF receptors, as well as small molecule anti- Antagonist drug. Agents may also be used in conjunction with radiation therapy or surgery.

Treatment with an agent of the present invention is not such an agent but the other is at least 30% or 40% of the median progression-free survival or overall survival time of a patient with cancer relative to an equivalent therapy, Can be increased by 50%, 60% to 70%, or even 100% or more. Alternatively, or alternatively, the treatment comprising the agent may be used to determine the complete response rate, partial response rate, or objective response rate (complete + partial) of a patient with a particularly relapsed or refractory cancer compared to the same treatment without a synbody At least 30% or 40%, preferably 50%, 60% to 70% or even 100%. Optionally, the treatment can inhibit tumor growth, invasion, metastasis or angiogenesis.

Typically, in addition to the agents of the present invention, in addition to the agents of the invention, a chemotherapeutic agent alone (or a placebo plus) is administered in clinical trials (e.g., Phase II, II / III or Phase III trials) The increase in median progression-free survival and / or response rate of the zero-treated patients is statistically significant at the p = 0.05 or 0.01 level. The complete and partial response rates are determined using objective criteria commonly used in clinical trials of cancer, such as those listed or approved by the National Cancer Institute and / or the Food and Drug Administration .

The effect of agonists on pain in humans can be assessed using a variety of tests. A number of pain questionnaires and scales were designed to assess patient pain using a variety of methods. Pain can be assessed on a single scale (strength alone) or multiple scales (duration and intensity). Useful pain scales include Visual Analog Scale, McGill Pain Questionnaire, and Descriptor Differential Scale (see, for example, J. Rheumatol. 9 (5): 768-9. PMID 6184474. Melzack. : 277-99, Gracely (1988), Pain 35 (3): 279-88). The patient's sensitivity to pain (pain threshold) may also be measured using a pain meter, such as a sonic palpometer, a pressure-controlled palmometer, a laser-based Dolorimeter Analgesia meter (IITC Life Sciences ), Baseline Algorimeter (Kom Kare Company), Bjornstrom pain system to measure skin sensitivity to pain, or Boas pain system to measure sensitivity on a mental area. Examples of drugs that can be co-administered for the treatment of pain include NSAIDs, COX-2 inhibitors, COX-3 inhibitors, iNOS inhibitors, PAR2 receptor antagonists, neuroleptic agents, opioids, N-acetylcholine receptor agonists, glycine antagonists , Vanilloid receptor antagonists, neurokinin antagonist calcitonin gene-related peptide antagonists and cyclooxygenase (COX) -suppressing nitric oxide donors (CINOD). Other pain relief medications include, but are not limited to, codeine, vicodin, morphine, Demerol, percocet, Darvon and Darvocet conotoxin, Symlin.

b) Prevention Methods

The present invention also provides methods and compositions for the prevention of said disorders in subjects at risk of disorders. Usually, such subjects have an increased risk of developing a disorder (e. G., A disease, disorder, disorder or disease) as compared to a control population. For example, the control population may include one or more objects that have not been diagnosed or have been randomly selected (e.g., matched by age, gender, household, and / or race) from a general population with a family history of the disorder have. The subject may be considered at risk for the disorder if a risk factor associated with the disorder is found to be associated with the subject. Risk factors may include any activity, characteristic, event, or characteristic associated with a given disorder, for example, through statistical or epidemiological studies in a population of subjects. Thus, the subject may be classified as at risk for the disorder even if the study identifying the underlying risk factors does not specifically include the subject. For example, subjects undergoing cardiac surgery have a risk of stroke because the incidence of transient cerebral ischemic attacks is increased in the population of subjects undergoing cardiac surgery compared to subjects without cardiac surgery.

Other common risk factors for stroke are age, family history, gender, previous incidence of stroke, transient ischemic attack or heart attack, hypertension, smoking, diabetes, carotid or other arterial disease, atrial fibrillation, Disease, heart failure, enlarged cardiac myopathy, heart valve disease and / or congenital heart defect; High blood cholesterol, and saturated fats, trans fats or high cholesterol.

Risk factors for cancer include genetic susceptibility to cancer, carcinogens, e.g., patients who have been exposed to radiation or toxins, and patients who have previously been treated for cancer and at risk of recurrence.

Risk factors for pain include surgery experience, exposure to combat or other hazards, or diseases associated with severe or chronic pain, such as diabetes and pain from cancer.

Agents that are optionally linked to an internalizing peptide are those of a patient (or animal model) at risk of the disease treated with the agent compared to a control group of a patient (or animal model) at risk of disease not treated with the chimeric agents of the invention Is a prophylactically effective therapy which refers to an amount, frequency and route sufficient to prevent, delay or inhibit the occurrence of one or more symptoms or signs of a disease in a subject, . The amount is also considered prophylactically effective if the individually treated patient achieves a favorable outcome over the average outcome in the control group of equivalent patients not treated by the methods of the present invention. Prophylactically effective therapies include the administration of a prophylactically effective amount by the frequency and route of administration necessary to achieve the intended purpose. For the prevention of stroke or other acute-onset diseases and disorders in patients at an urgent risk (e. G., Patients undergoing cardiac surgery), a single administration of the agent is usually sufficient. For long term risks, for example, in patients at risk for cancer following exposure to carcinogens, multiple administrations may be specified.

X. Pharmaceutical composition, dosage, and route of administration

The agent of the present invention optionally linked to the internalizing peptide can be administered in the form of a pharmaceutical composition. Pharmaceutical compositions are typically prepared under GMP conditions. The pharmaceutical composition may be presented in unit dosage form (i.e., a dose for single administration). The pharmaceutical compositions may be prepared by conventional mixing, dissolving, granulating, dragee-making, pulverizing, emulsifying, encapsulating, entrapping or lyophilizing processes. For example, lyophilized agents may be used in the formulations and compositions described below.

The pharmaceutical compositions may be formulated in conventional manner using one or more physiologically acceptable carriers, diluents, excipients or adjuvants to facilitate the treatment of the chimeric agents with the preparations which can be used pharmaceutically. Proper formulation depends on the route of administration chosen.

Administration may be parenterally, intravenously, orally, subcutaneously, intraarterially, intracranially, intrathecally, intraperitoneally, topically, intranasally, by inhalation, transdermal, e.g., transdermal, or intramuscularly via patch. Intravenous administration is preferred.

The pharmaceutical composition for parenteral administration is preferably a sterile and substantially isotonic pharmaceutical composition. For injection, the agent may be an aqueous solution, preferably a physiologically compatible buffer, for example, Hank's solution, Ringer's solution, or physiological saline or acetate buffer (unpleasant at the injection site ). &Lt; / RTI &gt; The solution may contain a formulating agent, for example, a suspending, stabilizing and / or dispersing agent.

Alternatively, the agent may be present in powder form for reconstitution with a suitable vehicle, e. G., Sterile pyrogen-free water, prior to use.

Agents of the invention may also be administered with other agents that increase the passage of agents of the invention across the blood brain barrier, such as mannitol, Tween® or DMSO.

For transmucosal administration, a suitable penetrator is used in the formulation for the barrier to be permeated. This route of administration may be used to deliver the compound to the nasal cavity or for sublingual administration.

For oral administration, the agent may be formulated with pharmaceutically acceptable carriers such as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc., for oral ingestion by the patient being treated. For oral solid formulations, for example powders, capsules and tablets, suitable excipients include fillers such as sugars such as lactose, sucrose, mannitol and sorbitol; Cellulose preparations such as corn starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methylcellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and / or polyvinyl Pyrrolidone (PVP); Granulating agents; And a binder. Upon demand, disintegrating agents such as crosslinked polyvinylpyrrolidone, agar, or alginic acid or its salts, such as sodium alginate, may be added. Upon demand, solid dosage forms can be sugar-coated or enteric-coated using standard techniques. For oral liquid preparations such as suspensions, elixirs and solutions, suitable carriers, excipients or diluents include water, glycols, oils, alcohols. In addition, flavoring agents, preservatives, coloring agents and the like may be added.

In addition to the formulations described above, the agent can also be formulated into a depot preparation. Such long acting formulations may be administered by implantation (e. G., Subcutaneous or intramuscular) or by intramuscular injection. Thus, for example, the compound may be formulated with a suitable polymer or hydrophobic material (for example as an emulsion in an acceptable oil) or an ion exchange resin, or with a poorly soluble derivative, such as a poorly soluble salt .

Alternatively, other pharmaceutical delivery systems may be used. Liposomes and emulsions can be used to deliver chimeric agents. Certain organic solvents, such as dimethylsulfoxide, may also be used, but are usually more toxic. In addition, the compound may be delivered using a semipermeable matrix of a solid polymer containing a sustained-release system, for example, a therapeutic agent.

Depending on their chemical nature, the sustained-release capsules may release chimeric agents for up to 100 days. Depending on the chemical nature and biological stability of the therapeutic reagents, additional methods for protein stabilization can be used.

Since the agents of the present invention may contain charged side chains or ends, they may be included in any of the formulations described above as free acids or free bases or pharmaceutically acceptable salts. A pharmaceutically acceptable salt is a salt which substantially retains the biological activity of the free base and is prepared by reaction with an inorganic acid. Pharmaceutical salts tend to be more soluble in aqueous or other protonic solvents than the corresponding free base forms.

The amount of the agent to be administered will depend on the patient being treated, the disease or disorder, whether the treatment is substantially therapeutic or prophylactic, the weight of the patient, the severity of the pain, the manner of administration, and the judgment of the prescribing physician. The treatment may be repeated intermittently even if the indication is detectable or even if the indication is not detectable. Treatment may be provided alone, or may be provided with other drugs.

Effective doses of the agents of the present invention can provide benefits without causing substantial toxicity. The toxicity of the agonist can be determined by standard pharmaceutical procedures in cell cultures or experimental animals and is determined, for example, by determining the LD50 (the dose lethal to 50% of the population) or the LD100 (the dose lethal to 100% of the population) . The dose ratio between toxic and therapeutic effects is the therapeutic index. Agents that exhibit a high therapeutic index, such as peptides or peptidomimetics, are preferred (see, for example, Finglmeat get ., 1975, In:The Pharmacological Basis of Therapeutics, Ch.1, p.1).

A chimeric agent comprising an internalizing peptide linked to an agonist may be used in the same or a smaller dose as the agent alone and may be administered by the same route as the pharmacological agent alone to treat the same disease (s) as the pharmacological agent alone &Lt; / RTI &gt;

A suitable dose of an agent optionally conjugated to the internalizing peptide is usually less than 25 mg / kg. The dose is sometimes in the range of 10-4 mg / kg to 25 mg / kg, for example 0.1 or 0.5 mg / kg to 1, 50 or 10 mg / kg. The total dose per patient may be calculated by multiplying the dose per kg of body weight by the patient's body weight in kilograms. For example, the total dose for a 75 kg patient can be calculated by multiplying the dose by 75.

XII . Screening method

One. Screened  agent

Agonists can be first screened in vitro for the desired binding or inhibitory activity. Agents may include ND2 peptides or peptidomimetics as described above. The agent to be screened can also be obtained from a library of natural sources, e. G., Marine microorganisms, algae, plants, fungi, or synthetic peptides or other compounds. Combination libraries that can be synthesized in a step-by-step manner can be generated for many types of compounds. Such compounds include polypeptides, beta-turn mimetics, polysaccharides, phospholipids, hormones, prostaglandins, steroids, aromatics, heterocyclic compounds, benzodiazepines, oligomer N-substituted glycines and oligocarbamates. Many combinatorial libraries of compounds are described in Affymax, WO 95/12608, Affymax, WO 93/06121, Columbia Umversity, WO 94/08051, Pharmacopeia, WO 95/35503 and Scripps, WO 95/30642 Which is incorporated herein by reference in its entirety for all purposes). Peptide libraries can also be generated by phage display methods. See, for example, Devlin, W0 91/18980.

2. In vitro  screen

Agents can be screened for their ability to bind Src peptides, including the desired binding activity, e. G., Src, or residues 40-49 of Src. Alternatively or additionally, the agent can be an ND2 or ND2 peptide as described above for its ability to bind to a peptide comprising Src or residues 40-49 or 40-58 thereof (e.g., residues 310-321 of ND2 &Lt; / RTI &gt; the peptide being constituted). Binding can be assessed by ELISA, fluorescence polarization, or Western blot in other ways. In some formats, one component of the association test is fixed. Various formats are possible as described in the embodiments. The ability of an agonist to bind Src and / or inhibit the binding of ND2 or ND2 peptides to Src is an indication that such agents have pharmacological activity useful in treating nervous system diseases, pain or cancer. Subsequently, the agonists can be further screened in various animal models as further described below.

Agents can also be screened for inhibitory activity against Src kinase. Kits for performing kinase assays are commercially available. Typically recombinantly expressed forms of Src are mixed with kinase buffers and peptides with tags that allow fixation in the presence of phosphorylatable residues and ATP. The amount of phosphorylated peptide that exhibits kinase activity can be detected using an antibody specific for the phosphorylated peptide. This assay is performed by the relevant Src activity in the presence and absence of agonist under test to determine whether the agent reduces phosphorylation.

3. Animal models of stroke

Agents can be tested in various animal models of stroke. In one such model, adult male Sprague-Dawley rats are subjected to transient middle cerebral artery occlusion (MCAO) for 90 minutes by the intraluminal suture method. Animals are fasted overnight, and atropine sulfate (0.5 mg / kg IP) is injected. After 10 minutes, anesthesia is induced. The rats are orally anesthetized, mechanically ventilated and paralyzed with pancuronium bromide (0.6 mg / kg IV). The body temperature is maintained at 36.5-37.5 ℃ using a heating lamp. A polyethylene catheter is used in the femoral artery and vein to continuously record blood pressure and to sample blood for gas and pH measurements. Transient MCAO was achieved for 90 minutes by the introduction of a poly-L-lysine-coated 3-0 monofilament nylon suture (Harvard Apparatus) into the circle of Willis through the carotid artery that effectively obstructed the middle cerebral artery . This causes a wide range of infarcts including the cerebral cortex and basal ganglia. Animals are treated with the agonist under study or with a negative or positive control. The treatment may be from 1 hour before to 1 hour after induction of ischemia. The negative control may be a vehicle. The positive control may be the Tat-NR2B9c peptide, YGRKKRRQRRRKLSSIESDV (SEQ ID NO: 6) previously identified as effective. After administration of the agent to the animal, the infarct volume and / or disorder index is determined. Infarct volume is usually determined after 24 hours of treatment, but can be determined at a later time, such as after 3, 7, 14 or 60 days. The impairment index can be monitored over time, e.g., 2 hours after treatment, 24 hours after treatment, 1 week and 1 month after treatment. Agents exhibiting a statistically significant reduction in infarct volume and / or disability index as compared to control animals not treated with agonists have been found to have useful activities in practicing the methods of the present invention.

Similar experiments can be performed on animals that have been subjected to permanent ischemia. Permanent ischemia of the middle cerebral artery smooth muscle vessels has been described in Forder et &lt; RTI ID = 0.0 &gt; al . , Am J Physiol Heart Circ Physiol 288: H1989-H1996 (2005). In short, PE 10 polyethylene perfusion is inserted into the right ECA. The skull is exposed through midline incision and two 6- to 8-mm windows are made on the right temporal cortex (5 mm lateral to 2 mm tail and lateral). The soft-tissue ganglia are visualized by injecting a small bolus (10-20 μl) of biotinylated blue-violet (10 mMol / L; Sigma) in normal saline into ECA. The same three soft-tissue carotid MCA branches are electrically cauterized and dye injection is repeated to ensure cessation of flow through the cauterized arterioles. Thereafter, the incision is closed, the animal is returned to her, and free access to food and water. This permanent ischemic model produces a very reproducible small infarct that is confined to the cortex based on the solidified terminal soft-gland artery.

4. Animal models of pain

Inflammatory tests on pain in mammals (e.g., rodents) were performed on a tail-flick (spinal-mediated refractory reflex) test (D'Amour et al . (1941), J. Pharmacol. Exp. Ther. 72: 74-79), a hot-plate test, a Randall-Selitto test (Swingle et al . (1971), Proc. Soc. exp. Biol. Med. 137: 536-538) and tail-pinch testing. The test can be used to determine the level of invasive thresholds for various types of noxious stimuli such as, for example, thresholds for heat (tail-stroke test, hot-plate test, Hargreaves' test of foot- Short immersion of hindpaw); Alternatively, for example, Von Frey test for allodynia test, J Neurosci Methods. 1999 Mar 1; 87 (2): 185-93). Dynamic harmless irritative pain can be assessed by tapping the soles of the soles of the hind paw using a swab, where dynamic innocuous irritative pain is considered present when the animal responds to the swab stimulus within 8 seconds of the start of the acupuncture . Pain responses to hazardous chemical agents can be monitored, for example, by monitoring abdominal twist after intraperitoneal injection of diluted acetic acid and by adding capsaicin to drinking water to test for aversive drinking test ). &Lt; / RTI &gt;

Tests for inflammatory pain and hypersensitivity were made using formalin paw test (Tjolsen et &lt; RTI ID = 0.0 &gt; al . (1992), Pain 51: 5-17), complete Freund's Adjuvant paw test (CFA), test for formalin-induced facial pain (Clavelou et al . (1989), Neurosci. Lett. 103: 349-353), and foot tests following administration of a substance such as carageenan, capsaicin or bradykinin. Arthritic diseases can be stimulated by a variety of models including agonists such as carrageenan, uric acid or mustard oil, or injection into various joints of the adjuvant. Visceral pain can be modeled by intraperitoneal injection of substances such as bradykinin, acetylcholine, acetic acid or phenylquinone. Streptozocin (STZ) -induced diabetic neuropathy model induces reproducible mechanical innocuous stimulation pain within 3 weeks (Chen and Pan, J Neurophysiol 87: 2726-2733, 2002).

Tests for neuropathic pain resulting from peripheral nerve injury include chronic contraction injury (see, for example, Bennet and Xie model of sciatic nerve ligation, Pain 33: 87-107); Partial neural ligation (Seltzer et al . , J Basic Clin. Physiol. Pharmacol. 1991), spinal nerve interaction or ligature (Lombard et &lt; RTI ID = 0.0 &gt; al . , Pain. 1979 6: 163-74; Kim & Chung, Pain. 1992; 50: 355-63), cryoneurolysis (Deleo et al . , Pain. 1994; 56: 9-16), sciatic nerve ischemia (Kupers et al . , Pain. 1998; 76: 45-59). A typical test is the tactile harmless stimulation pain test (Chaplan et &lt; RTI ID = 0.0 &gt; al . (1994) J. Neurosci. Meth. 53: 55-63). Taxol-induced neuropathic pain does not contain an inflammatory component. Specific models for specific peripheral neuropathy diseases include trigeminal neuralgia (Vos and Maciewicz, J Neurosci. 1994; 14: 2708-23), diabetic neuropathy (Burchiel et al . , Diabetes. 1985; 34: 1210-3), and vincristine neuropathy (Aley et al . , Neuroscience. 1996; 73: 259-65). Neural model (Wall et al . , Pain. 1979 Oct; 7: 103-11) can reflect the emptying tube resulting from truncation.

Animal models of pain resulting from spinal cord injury include cord interactions or partial interactions (Levitt & Levitt, Pain. 1981; 10: 129-47), stimulation-induced ischemia models (Hao et al . Neurosci Lett. 1991, 8; 128: 105-8), excitotoxic model using the injection of the spinal cord quinolyl seukwal rate (quisqualate) (Yezeierski & Park, Neurosci Lett 1993 9; 1571: 115-9) and contusion model (Siddall et al . , Neuroreport. 1995; 6: 1241-4).

5. Animal models of epilepsy:

A large number of animal models of various epilepsy disorders have been widely characterized. See, for example, Models of Seizures and Epilepsy (ed. Pitkanen et &lt; RTI ID = 0.0 &gt; al . , ISBN: 978-0-12-088554-1; Elsevier Inc., 2006). The animal may vary from fruit flies to primates, where epilepsy occurs in a variety of ways, including the application of chemical or genetic screening to spontaneously onset seizures and / or epilepsy. Examples of animal models include, but are not limited to, high-fever-induced seizures in rats mimicking heat-excitation, mouse mutations such as totterer, stargazer, lethargic, and short- Slow wave epilepsy (SWE) mice that share features similar to human absence epilepsy, such as the human epilepsy (e. G.) Or gazing).

An animal model that has been widely characterized for multiple partial seizures observed in patients with temporal lobe epilepsy (TLE) has also been described. The kininic and pilocarpine (PILO) seizure models will be the most commonly studied compound-induced animal models for TLE. Kindling, a phenomenon caused by repetitive topical application of electrical stimulation of the first quasi-seizure eventually leads to strong partial and systemic seizures, which is a beneficial model for TLE.

In addition, several animal genetically susceptible epilepsy species have been described as animal models for studying photosensitive and auditory nerve reflex epilepsy. These include Papio papio, Fayoumi epilepsy (FEpi) strains of chicken, genetically susceptible rat (GEPR) and DBA / 2 mice.

A variety of methods are available to induce systemic rigidity-to-seizure or non-seizure activity in animals, which is also true for some genetic animal models that are susceptible to seizures or have spontaneous seizures. The following are a few traditional methods for deriving the type of seizure.

Spastic seizures characterized by rigid hindlimb extension / post-bending mobility are reliably induced by maximum electrical shock, a continuously popular method for rapid screening of new anticonvulsant drugs.

Pentylenetetrazole (PTZ) is the most widely used systemically administered seizure inducer. Repeated injections of PTZ can be provided to generate the types of electrical ignition and ignition chemical ignition. At high doses, PTZ (usually subcutaneously or intravenously administered) certainly induces stiff-to-spastic seizures in rats or mice, which is a rapid and effective measure of both seizure susceptibility and screening of new drugs. PTZ can also be used to induce spalling-like seizures when delivered at low doses throughout the body.

Plutyryl, hexafluorinated ethers are chemical inhalants used to induce reproducible seizure patterns in rodents. In this way, the rat or mouse is placed in a confidential chamber where the parenteral flu shot is dispensed, and after 10-20 minutes, the flurothyl first causes basilar traumatic spasm, followed by severe seizure- &Lt; / RTI &gt; Finally, another experimental animal model for systemic necrosis is the rat pancreas (GAERS, WAG / Rij, &lt; RTI ID = 0.0 &gt; SER) and a number of genetic models in mice (stargazer, tottering, somnolence, slow-wave epileptic mice, mocha, and ducky).

Animal models such as those described above both in vivo and in vitro are valuable in understanding the basic mechanisms of partial or systemic seizure-related phenomena and are standard techniques for evaluating new therapeutic agents. See Sarkisian, Epilepsy & Behavior 2, 201-216 (2001), which is incorporated by reference in its entirety.

6. Animal models of anxiety

Anxiety can be induced by placing an animal, e. G., A rat, in an unfamiliar environment and observing the response (e. G., Crossing a line grid or selecting an open or closed tube). For example, a rat can be tested in an open field stage to determine both the arousal state and the ability to become accustomed to the new environment, and can be evaluated from the intersecting grid lines. Decrease in crossing indicates reduced anxiety. Rats may also be tested in the maze to assess anxiety / emotional in the rat. The appropriate maze has four arms (two open, two closed: 15 cm wide and 60 cm long) extending from the center platform and raised 1.5 m from the floor. The rats are placed in the center of the maze, provided with a free choice to allow entry into any arm, and entry is operationally defined as entry of the head and forelegs into the arm. The time spent in the open or closed arm is recorded and scored from simultaneous video recording from two directions (head and horizontal). An increase in time spent in an open arm indicates decreased anxiety because the rats tend to avoid the open space by nature.

7. Animal models of cancer

The activity of the agonist against cancer can be tested in immunodeficient mice or rats transplanted with human tumors. Examples of immunodeficient lines of mice that may be used include nude mice, e.g., CD-1 nude, Nu / Nu, Balb / c nude, NIH-III (NIH-bg-nu-xid BR); scid mice, for example, Fox Chase SCID (CB-17 SCID), Fox Chase random mating SCID and SCID Beige; Mice deficient in RAG enzyme; It is also a nude rat. Experiments are described, for example, in Kim et &lt; RTI ID = 0.0 &gt; al . , Nature 362: 841 (1992). Human tumor cells normally grown in complete DMEM medium are typically harvested from HBSS. Female immunodeficiency, e. G., 5x10 &lt; ⁶ &gt; cells in 0.2 ml HBSS is subcutaneously injected into the equine area of athymic nude mice (4-6 weeks old). When the tumor size reaches 50-100 mm ³ , the mice are grouped at random and the appropriate agent therapy is applied at the same time as the contrast agent-deficient contrast therapy. Tumor size is usually determined twice a week by measuring two dimensions (length (a) and width (b)). Tumor volume is calculated according to V = ab2 / 2 and is expressed as mean tumor volume ± SEM. The effect of agonists can be measured by tumor growth over time, prolongation of survival of the mice, or increase of the percentage of mice that survive indefinitely or for a given time. A statistical analysis relative to the control can be performed, for example, using a Student's t test.

8. Internalization Peptides

Peptides or other agonists can be tested for internalization or transport activity in animals. Agents (e. G., Tat peptides) are, for example, labeled and can be injected into an animal, e. For example, intraperitoneal or intravenous injection is suitable. After approximately one hour of injection, the mice are sacrificed and perfused with fixative (3% paraformaldehyde, 0.25% glutaraldehyde, 10% sucrose, 10 U / ml heparin in saline). Thereafter, the brain is separated, frozen, and flaked. The flakes are analyzed for fluorescence using a confocal microscope. The internalizing activity is optionally determined from fluorescence relative to positive and negative controls. A suitable positive control is an agonist comprising a standard Tat peptide. A suitable negative control is a fluorescently labeled activator deficient in Tat. An unlabeled vehicle can also be used as a negative control.

Similar experiments can be performed in cell cultures to test internalizing peptides or other agonists (see US20030050243). A variety of fluorescently labeled Tat peptides, optionally linked to active peptides, are applied to cortical nerve cultures. After application, absorption is determined using a fluorescence microscope over several minutes. Increased absorption may be determined relative to positive and negative controls as described for experiments on absorption in animals.

Example

Example # 1: Identification of the ND2 sequence responsible for binding to Src kinase

GST-fusion proteins were designed using various fragments of ND2, expressed using standard protocols, and purified. These purified proteins were spotted onto membranes for probing with biotinylated Src 40-58 peptides or scrambled control peptides (sSrc 40-58). Typically 1-10 g of peptide or recombinant protein was spotted onto nitrocellulose and allowed to dry overnight. The membranes were blocked with 5% milk at room temperature for 1 hour, then incubated with biotinylated peptide (~ 15 [mu] g / ml) for 2 hours and washed with standard wash buffer. Bound probes were detected using a short incubation with streptavidin conjugated to horseradish peroxidase (SA-HRP) and a standard detection reagent of predominantly chemiluminescent kits. 1B shows the construct of the first set, Fig. 1C shows that the sub-fragment ND2.1 (AA239-321), which can bind to Src 40-58, also binds to Src 40-58 Dot blot. Additional GST-constructs were prepared to narrow the core Src binding region (Figure 2A). The constructs were used to make dot blots and tested for their ability to capture biotinylated Src 40-58 (Figure 2B). While ND2.1.4 (AA 289-321) was the most effective subfraction for Src 40-58 binding, none of the fragments tested bound to the scrambled negative control (B-sSRC 40-58) Specificity of action.

As confirmation, an ELISA assay was performed to verify the binding in various assay formats. GST-ND2, GST-ND2.1 or GST-ND2.1.4 were coated onto ELISA wells by incubation at the indicated concentrations under standard conditions. The wells were blocked with 5% milk and incubated with biotin-Src 40-58 (top of Fig. 2C) or biotin-sSc 40-58 (bottom) with 6 uM. SA-HRP and standard detection reagents showed that Src 40-58 was able to bind to each of the ND2 constructs in a concentration-dependent manner.

Additional GST constructs were prepared to identify the amino acid sequence at ND2.1.4 responsible for binding to Src 40-58 (Figure 3A). Although the sequence minimally binds, the sequences flanked by the ND2 sequences as well as the sequences corresponding thereto can be used to inhibit the Src-ND2 interaction. Dot blots probed with Biotin-Src 40-58 using the GST protein showed ND2 310-321 binding with high relative affinity. In addition, binding was observed using ND2, ND2 289-309, 299-318, 302-321, and 307-321 (FIG. 3B). As described above, the interaction with all of the fragments was confirmed by ELISA assay (Fig. 3C) and the best combinations were ND2 310-321, ND2 289-309, ND2 299-318, ND2 307-321 and full length ND2. All four fragments provided superior binding to Src 40-58 over full length ND2. All fragments tested were able to bind to Src 40-58 more strongly than the negative control peptides sSrc 40-58.

We next tested the ability of the ND2 construct to bind to the shorter form of the Src binding domain-Src 40-49 (Fig. 4A, B). ND2 310-321, ND2 307-318, and ND2 310-318 were the best combinations of Src 40-49 by dot blot analysis (Fig. 4A). These interactions were confirmed by ELISA and ND2 310-321 showed the strongest binding.

Next, the present inventors confirmed the interaction between ND2 310-321 and Src 40-49 in various assay formats using various constructs. Figure 5A shows dot blots probed with biotinylated ND2 310-321 (Bio-ND2 310-321) or scrambled biotinylated ND2 310-321 (Bio-sND2 310-321) . Src 40-49-Tat represents a fusion peptide fused to the C-terminus of the Src 40-49 sequence to generate a peptide having the human HIV-1 tat protein transduction domain [YGRKKRRQRRR] having the sequence KPASADGHRGYGRKKRRQRRR, while Tat-Src 40-49 represents a fusion peptide in which the Tat domain is fused to the N-terminus of Src 40-49 to generate a peptide having the sequence YGRKKRRQRRRKPASADGHRG. The dot blot shows that both Tat fused Src 40-49 peptides were bound to ND2 310-321 as did the Src 40-49 peptide itself. Binding of biotinylated ND2 310-321 to the labeled plated Src peptides was also evaluated in an ELISA assay, and each of the four Src constructs was able to capture biotin-ND2 310-321 (Fig. 5B). The peptide was plated at a concentration of ~ 5 μM. Similarly, when ELISA plates were coated with Tat-ND2 310-321 under standard conditions, bio-Src 40-49 was able to bind in a concentration dependent manner, whereas with biotinylated scrambled control peptides, (Fig. 5C; Bio-sSc 40-49).

Competitive ELISA was also performed. Figure 5D shows a competitive ELISA assay testing the ability of tat Src 40-49 to inhibit the binding of biotinylated Src 40-49 to ND2 310-321. The results show that the binding of biotinylated Src 40-49 to ND2 310-321 was inhibited by the tat Src 40-49 peptide. Figure 5E shows the ability of Src 40-49-Tat to inhibit the binding of biotinylated Src 40-49 to Tat-ND2 310-321 previously coated on ELISA plates. The results show that the binding of biotinylated Src 40-49 to Tat-ND2 310-321 was inhibited by the Src 40-49-Tat peptide. In a similar manner, an ND2 construct with an amino acid sequence from the region 300-321 can serve as an inhibitor of the interaction between ND2 and Src.

Figure 6 also shows that Tat-ND2 310-321 can bind to Src 40-58 better than GST-ND2. Figure 7 shows that increasing the concentration of Tat-ND2 310-321 can compete with binding of Bio-Src 40-49 to the coated ND2 310-321 peptide in competition ELSIA.

Overall, the core ND2 sequence mediating the binding between ND2 and Src is likely to be present between amino acids 310 to 321 of ND2, and the amino acid sequence of 289 to 321 is likely to contribute to or affect the binding of ND2 and Src.

Example  #2: In vivo SRC - ND2 of Joint localization  However, ND2  And NMDAR Lt; RTI ID = 0.0 &gt; localization &lt; / RTI & SRC - ND2  Inhibitor of interaction

A series of experiments were performed to test the localization of ND2, Src, NMDAR subunits, and PSD95 in hippocampal neurons by immunocytochemistry in the presence of Tat, Tat-ND2 310-321, or Src 40-49-Tat . Briefly, hippocampal neurons were isolated from fetal E18 Wistar rats at day 17 and cultured in neurobasal media containing coverslip with B27 and glutamax. The cells were then treated with 1 [mu] M peptide for 1 hour and fixed by standard methods. Proteins were visualized using secondary antibodies coupled to specific antibodies and fluorescent molecules. Combine the images into Photoshop and calculate the localized% localized between the pairs as the total co-localized fluorescence colonies divided by the total number of clusters (eg% of ND2 co-localized with Src = Localized total) / (total ND2 colonies); co-localized Src% = (co-localized total) / (total Src colonies) with ND2.

Figures 8A and 8B show that incubation with Tat-ND2 310-321 or Src 40-49-Tat can reduce the amount of co-localization of Src and ND2, while the control Tat carrier is not. Thus, such peptides can cross cell membranes, disrupt preformed complexes in cells, and can be used as therapeutic agents.

Next, joint localization of ND2 and NMDA receptor subunits 2B (NR2B) and PSD95 in the presence of the inhibitor was tested. Both Tat-ND2 310-321 and Src 40-49-Tat disrupt the interaction between ND2 and Src but do not significantly affect the association between ND2 and NR2B (Figure 8A, B) 9A, B). Surprisingly, the disruption of the interaction between Src and ND2 reduces the co-localization of ND2 and PSD95 (Fig. 9C, D). Since PSD95 is known to associate with NR2B through interaction between the C-terminus of NR2B and the first two PDZ domains of PSD95 (Aarts et al., Science, 2002), the drug disrupts the NMDAR-PSD95 interaction And methods of achieving the advantage of disrupting the NMDAR-PSD95 interaction. These include treatments for stroke, excitotoxicity-related disorders, pain, neurodegenerative disorders, anxiety, epilepsy, optic neuropathy, and the like. Consistent with the collapse of the co-localization of PSD 95, Figures 9E and F show that the co-localization between PSD 95 and NR2B similarly collapses. In a similar manner, Tat-ND2 310-321 and Src 40-49-Tat can reduce co-localization between NR2B and Src (FIG. 10) and PSD95 and Src (FIG. 11).

Thus, compounds containing ND2 sequences that inhibit binding between Src and ND2 can modulate the association between NMDA receptor complexes, particularly NR2B and Src, and between NR2B and PSD95.

Antibody

The following primary antibodies were used in this study: mouse mAbs (Ca #: ab28373) for NR2B, mouse mAbs for clone 7E3-1B8, Cat #: ab13552 for PSD95 and mouse mAbs for clone 327, Cat # : ab16885) was purchased from Abcam (Cambridge, Mass.); Rat anti-NR2B (Cat #: 06-600) was purchased from Millipore (Temecula, CA); (Z-5, Cat #: sc-459) were purchased from Santa Cruz Biotechnology (Santa Cruz, Calif.); (Tyr 1472, Cat #: 4208) and rabbit anti-PSD 95 (D27E11, Cat #: 3450) were transfected with Cell Signaling Technology ( Danvers, Mass.).

All of the following secondary antibodies used in this study were purchased from Jackson ImmunoResearch Laboratory (Weat Grove, PA): Texas Red - donkey harbor - rabbit (711-075-152), Texas red donkey harbor - mouse (711-075-150), Texas Red-donkey anti-goat (705-075-003), FITC-donkey anti-rabbit (711-095-152), FITC-donkey anti- mouse (715-095-150) , FITC-donkey anti-goat (705-095-003), peroxidase goat anti-mouse (115-036-006), peroxidase goat anti-rabbit (111-036-047) and peroxidase rabbit anti- - Chlorine (305-036-003).

Immune cell chemistry

The hippocampal neurons cultured in cover slips were treated with 1 μM Src 40-49-Tat, Tat-ND2 310-321 or Tat for 14 days to 1 hour. The neurons were then incubated for 10 minutes in 4% paraformaldehyde and 4% sucrose in phosphate-buffered saline (PBS), and then at room temperature (RT) for 5 minutes in 20% methanol. Cells were permeabilized with 0.25% Triton X-100 in PBS for 5 min and treated with 5% donkey serum in PBS for 30 min at room temperature. The culture was incubated for 2 hours at room temperature with a mixture of primary antibodies generated in various species in 0.25% Triton X-100 PBS, washed, and seed-specific for all species generated in donkeys and conjugated to Texas Red or FITC fluorophore Was incubated with a mixture of secondary antibodies (1: 200 dilution in 0.25% Triton X-100 PBS) for 1 hour at room temperature. The cover slips were washed with PBS and mounted with ProLong Gold antifade reagent (Invitrogen). Images were collected on a Nikon Eclipse TE 200 microscope using a 60 × pan-fluor objective. Images were analyzed with PhotoShop 7 (Adobe, San Jose, CA). Adjust brightness and contrast, sharpen with an unsharpen mask tool, and combine the images for color co-localization.

For quantification of co-localization, the maximum intensity of the fluorescence short channel was standardized, the background fluorescence of each channel observed in the dendrite was subtracted, and the color images were combined. Two colonies of two different channels were considered as co-localization when at least 66% of the surface of one colony in one of the two channels overlap with the colony of another channel. For each combination of antibodies, three independent immunofluorescence experiments were performed. Each measurement was performed from a 50-m-long dendrite segment (having an average width of 2 m). Joint localization was expressed as a percentage of the total colonies analyzed.

Example # 3: Inhibitor of ND2-SRC interaction affecting composition of NMDA receptor complex

Co-immunoprecipitation experiments were used to test the status of selected proteins in NMDA receptor complexes in neurons. This was tested in the presence or absence of inhibitors of Src-ND2 interaction in both hippocampal neurons and rat brains applied to stroke using the 3 PIAL vascular occlusion model (3PVO). Figures 12A-E show the status of NMDAR in hippocampal neurons of 14 days. Antibodies against ND2, Src, PSD95, NR2B, and IgG alone were added to the lysates as described below and used to generate clean immunoprecipitates (antibodies listed at the top of the blot). Each blot was then probed with a detection antibody using standard Western Blot techniques using the antibodies listed below each blot. This figure shows that each of the immunoprecipitated antibodies can pull down the complex containing ND2, Src, PSD95 and NR2B.

We next tested the effect of Tat-ND2 310-321 and Src 40-49-Tat on association of proteins in the NMDAR signaling complex in hippocampal neurons cultured in 14DIV. Neurons were treated with either Tat-ND2 310-321 or Src 40-49 at 1 μM (Figure 13A) for 1 hour or 3 μM (Figure 13B) for 2 hours. Cell lysates were purified and immuno-precipitated with anti-NR2B (left) and then stained with anti-NR2B, anti-PSD95, anti-Src and anti-ND2 antibodies. These studies indicate that treatment of neurons with both Tat-peptides reduces the association of NR2B and Src. At the concentrations and exposure times used, the association of PSD95 and NR2B was slightly reduced when pretreated with Tat-ND2 310-321. This experiment was repeated, indicating that the Tat-ND2 310-321 treatment significantly reduced the amount of PSD-95 and Src in the NMDAR complex when compared to Src 40-49-Tat after immunoprecipitation with antibody to NR2B (Fig. 13C, left). When lysates treated with the same peptide were used for immunoprecipitation with anti-PSD95 antibody, treatment with Tat ND2 310-321 significantly reduced the amount of NR2B normally associated with PSD95. In addition, in another set of immunoprecipitation experiments with anti-PSD95 antibodies against peptide-treated hippocampal neuron lysates, Tat-ND2 310-321 was able to nearly abolish association between NR2B and PSD-95, and PSD95 Lt; RTI ID = 0.0 &gt; (FIG. 13D). &Lt; / RTI &gt;

Next, the state of the protein was tested in rat brain applied to 3PVO stroke. After 3PVO ischemia, rodents were administered Tat-ND2 310-321 or saline via intravenous injection of the tail. One hour after surgery, the brain was rapidly harvested and lysed. Following IP with anti-Nr2B antibody, membranes were incubated with anti-phosphotyrosine antibody, developed and probed with anti-Src, phosphorylated Src (pTys) and anti-PSD95 antibodies. As an internal control, the ipsilateral and contralateral hemispheres (I and C, respectively) were prepared. Figure 15 shows that, in the Tat-ND2 310-321 treated animals, the PSD95 with the NR2B subunit is much less immunoprecipitated and the amount of Src is also reduced. This change in the NR2B composition appears to occur only in the hemisphere of stroke, as PSD95 appears to remain associated with NR2B in hemispheres not applied to stroke. This is significant because the reduction of PSD95: NR2B association in stroke tissue is protective but the NMDAR complex is required for other tissue functions such as neurotransmission and long-term potentiation. Thus, Tat-ND2 310-321 has the potential to selectively protect brain regions affected by stroke without disrupting the complex in non-diseased areas, suggesting that side effects may be reduced when compared to systemic PSD95: NMDAR inhibitors This is big.

A second set of experiments in which saline, Src 40-49-Tat or Tat-ND2 310-321 were modified to be administered by tail vein 1 hour after surgery was performed in animals treated with 3PVO, the brain was harvested, Lt; / RTI &gt; Figures 16A and B show the results of immunoprecipitation from the brain using PSD95 (Figure 16A) or NR2B (Figure 16B) as IP antibodies. In A, Tat-ND2 310-321 markedly reduced the amount of NR2B, phosphorylated NR2B and Src associated with PSD95. Conversely, in B after immunoprecipitation with the anti-Nr2B antibody, very little PSD95 was associated with the complex, and Src and PhosphoSrc (pSrc) were also less associated. The Src 40-49-Tat construct was not nearly as effective in mating PSD95 and Src from NMDA receptors.

Co-immunoprecipitation method

NMDAR and its associated proteins were prepared from cultured hippocampal (HP) neurons or rat brain. For in vitro competition experiments, HP neurons were harvested after 1 week of treatment with 1 μM concentration or 2 weeks with treatment with Src 40-49-Tat, Tat-ND2 310-321 or Tat peptide for 2 hours at 3 μM concentration, Were quickly collected and homogenized. For in vivo experiments, 1 hour post 3PVO ischemia, the rat was injected with the indicated peptide or saline by intravenous tail vein. After 2 hours of operation (1 hour after administration of the compound), the selected cortex of the brain was rapidly harvested and homogenized.

The lysates were incubated with Dynabeads protein G (Invitrogen) and selected antibodies for 30 minutes at room temperature. The separated immunoprecipitates were dissolved using SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was probed with the selected detection antibody, then stripped and re-probed with the other detection antibody.

Example # 4: ND2 inhibitor inhibiting PACAP-enhanced NMDA-induced current in CA1 neurons

The pituitary adenylate cyclase-activating polypeptide (PACAP) selectively increases NMDA receptor mediated responses in rat neurons, and these peptides are associated with synaptic plasticity as well as with modulation of both long-term potentiation and degradation . To test the effect of ND2 310-321 and Src 40-49 on PACAP-enhanced NMDA-induced currents, isolated CA1 neurons were subjected to a series of patch clamp experiments in the presence or absence of the peptides. Figure 14A shows the peak current normalized to PACAP (1 nM) or PACAP + ND2 310-321 (6 mM) in a patch pipette. ND2 310-321 can prevent PACAP-induced enhancement. Similar experiments were also performed using the Src-selective inhibitory peptide, Src (40-58) (14 mM), or the truncated peptide, Src (40-49) (6 mM). The reaction before application of PACAP was normalized to the reaction after 25-30 minutes of application of PACAP. The bar graph represents the recording of three groups from neurons and shows the relative change in peak NMDAR current under three conditions. ND2 310-321 inhibited PACAP-enhanced NMDA-induced current, whereas Src 40-49 did not. Thus, ND2 310-321 may be used to block PACAP enhanced NMDA-induced signaling, and thus may be useful for the treatment of many neurological diseases and disorders associated with impaired long-term potentiation (e.g., aging, Alzheimer's disease, Neurological disorders affecting memory).

Example  # 5: Reducing pain sensitivity in rodents ND2  Inhibitor

In order to demonstrate that Tat-ND2 310-321 is effective in reducing pain hypersensitivity, 10 pmol / g of Tat ND2 310-321 or scrambled ND2 or Src negative control peptide is injected from subcutaneous injection of complete Freund's adjuvant (CFA) Were administered via tail vein injection to rats with previously induced inflammation in the hind paws of rats. Measurements of pain hypersensitivity were performed at 1 hour or 2 hours after peptide injection using various stiff filaments as described in the method above. Animals treated with Tat-ND2 310-321 were significantly less sensitive to pain than animals treated with scrambled control peptides. In addition, peptides containing the ND2 310-321 region, such as tat-ND2 310-321, performed significantly better than Src 40-49-Tat in this experiment. This difference was greater after 1 hour of administration than 2 hours after administration under the conditions (FIG. 17).

Src 40-49-Tat has been reported to disrupt the association of Src and ND2 in nerve lysates (Liu, XJ et al . , (2008) Nature Medicine, References 3). This disruption also reduced association between the Src and NMDA R2 subunits, in turn decreasing the NMDA current and has beneficial effects on both inflammatory pain (formalin and CFA models) and neuropathic pain (peripheral nerve contraction). This result demonstrates that ND2 310-321 has excellent ability to provide pain relief in the CFA model (Figure 17), better disrupting associations between Src and NMDA R2 subunits (Figure 13C-Tat-ND2 or Src 40- (Probed with IP NR2B, anti-Src in the presence of 49-Tat) and decreased NMDA current (Fig. 14B). The present inventors have found that the ND2 compounds containing the 310-321 region of ND2, such as Tat-ND2 310-321, are capable of inhibiting the expression of Src 40-49- Acting better than Tat, and has the potential to become an effective therapeutic for the treatment of pain associated with inflammation and neuropathic pain, chronic pain, and irritability.

Way

CFA model:

Animals : Sprague-dolly rats (male, 250-300 g) from Charles River Laboratories (St. Constant, Quebec) were tested. They were bred in a plastisphere containing 1/8 "corn cob litter and kept in a 12-hour name / cancer cycle. They were bred in pairs and allowed an unlimited approach to food and water. The use of animals was in accordance with the guidelines of the Canadian Council on Animal Care and was approved by the Animal Care Committee at Toronto Western Hospital.

Induction of inflammation : Briefly, complete Freund's adjuvant (CFA; Sigma Chemical Company, St. Louis) was subcutaneously injected (100 L, 27 G½ needle under isoflurane anesthesia) on the plantar surface of the hind paw. The animals were returned to their teeth for 8 hours, causing inflammation and sensitization.

Tail intravenous : To administer the peptide intravenously after induction of inflammation, the animal was placed in a clear induction chamber and anesthetized with a mixture of 2.5% isoflurane in oxygen until the animal was completely anesthetized (after about 1 minute). The mask was then placed on the animal's nose and mouth and the isoflurane concentration was lowered to 1.5-2.0% during the injection period. The peptides were dissolved in 1 [mu] l / g of sterile saline and injected with 25 G [1/2] butterfly needles (Fisher Scientific). Later, animals were returned to us.

Measurement of Foot Avoidance Threshold : Pitcher et al. (Journal of Neuroscience Methods, 1999), the animals were placed on a platform containing 1.5 mm diameter holes in a clear plexiglass observation chamber (30 x 30 x 30 cm). A series of elevated von Frey filaments (Stoelting, USA) (. 008g-15g) was applied to the plantar surface of the hind paw to determine the minimum stimulus required to induce foot evasion. Each filament was applied vertically for 2 seconds or until evasion occurred. Each successive plenum was applied five times at 5 second intervals. Thresholds were determined by the filament that caused 3 positive reactions during 5 applications. No filaments larger than 15g were used to avoid tissue damage. Foot thresholds were measured before CFA injection, after 8 hours of CFA injection, after 60 minutes of peptide injection and 120 minutes after peptide injection.

Example # 6: ND2 inhibitor effective to reduce brain damage after stroke

The efficacy of ND2 inhibitors in the treatment of stroke was tested using three models of soft-vascular occlusion (3PVO) of stroke. This is a permanent model of stroke that causes a small but stable infarct at 24 hours.

Male Sprague Dawley rats (n = 10 in each group) were surgically applied to the permanent occlusion of the soft-tissue vasculature. One hour after surgery, the animals were given an intravenous injection of saline, Tat-NR2B9c (Aarts et al., Science, 2002), Src 40-49-Tat or Tat-ND2 310-321, Restored. Twenty-four hours after surgery, the brain was harvested, spliced, and incubated with TTC to visualize the infarct area. The infarct volume of each animal, as well as the average volume for each group of 10 were determined. Figure 18 shows the results of the above experiment. Both Tat-ND2 310-321 and Tat-NR2B9c showed about 40% reduction in infarct volume whereas Src 40-49-Tat was not statistically effective in reducing the infarct volume in the model. This suggests that the ND2 inhibitor of the Src-ND2 interaction, particularly Tat-ND2 310-321, may be an effective drug for the treatment of stroke.

In a second experiment, the efficacy of the estrastated forms of the two ND2 peptides was evaluated relative to Tat-ND2. The sequence is provided in the following examples. Both peptides were stistylated via an amide attached to the alpha-amino group of the N-terminal amino acid of the peptide. Myr2-ND2 provided equivalent or better protection against damage from stroke in this model, and myr-ND2 was less effective (Fig. 22). Thus, both Tat-ND2 and myr2-ND2 are effective drugs for the treatment of stroke when administered after stroke. The inventors have also found that the inhibitor is effective when administered prior to stroke.

Three ischemic soft-vascular occlusion models

Male Sprague Dawley rats (n = 10 in each group) weighing 230 to 290 g were used in this study. Experiments were performed in fasted rats (free access to water overnight, but not food). Three permanent soft-tissue vascular occlusions (3PVO) were previously described in Forder J et &lt; RTI ID = 0.0 &gt; al . , &Lt; / RTI &gt; 2005, supra). Briefly, rats were inoculated with 0.5 ml / kg of ketamine (100 mg / kg), acepromazine (2 mg / kg), and xylazine (5 mg / kg) Anesthetized with injection. An anal temperature probe was inserted and the animal was placed on a heating pad maintained at 37 &lt; 0 &gt; C. The skull was exposed through an intermediate incision and the tissue was scraped off. Using a dissecting microscope and an air-borne dental drill, the right body sensory cortex (5 mm lateral to the 2 mm tail and the bregma) was obtained by drilling a rectangle through the skull while keeping the hard membrane intact, Made two 6- or 8-mm windows. Three soft - wall arteriovenous middle cerebral artery branches around the barrel cortex were selected and electrically cauterized through the hard membrane. After the procedure, the scalp was sutured. Each rat was returned to its respective individual under a heating lamp to maintain body temperature until the rat was fully recovered. After 1 hour of 3PVO ischemia, the rat was injected with the drug (3 nmol / g) or saline indicated by intravenous tail vein. Food and water. Twenty-four hours after surgery, the brain was rapidly harvested. Tubular slices (2 mm) were collected through the brain and incubated in 2% triphenyltetrazolium chloride (TTC) (Sigma) for 15 minutes at 37 ° C. The image was scanned (Canon 4200F).

Example  # 7: Reduced pain hypersensitivity in a rodent model of neuropathic pain ND2  Inhibitor

The present inventors have used Tat-ND2 (Neuropathic pain) for neuropathic and inflammatory pain behavior using a model of peripheral nerve injury (PNI) and a complete Freund's adjuvant model (CFA) both of which are characterized by a reduction in mechanical foot- The effect of the scrambled D2 negative control (sTat-ND2 310-321) on the 310-321 control group was tested. The effect of Tat-ND2 310-321 on mechanical avoidance threshold was evaluated at 8-15d after PNI. The present inventors found that when Tat-ND2 310-321 was administered intravenously, the nerve injury caused a significant increase in foot-phobic threshold in the ipsilateral side, whereas sTat-ND2 310-321 did not. Similar results were observed in the CFA model (Fig. 17), where an increase in foot evasion threshold occurred within the first 45 minutes and lasted over the 3 hour trial period.

Thus, Tat-ND2 310-321, and peptides or peptidomimetics containing these regions of ND2 can provide relief from neuropathic pain.

Way

Chronic sciatic nerve contraction model : Animals: To generate peripheral nerve injury (PNI), a 2 mm polyethylene cuff was implanted surgically around the sciatic nerve of rat or mouse under isoflurane anesthesia. Animals were acclimated for 7 days before testing.

Drug administration : Tat-ND2 310-321 or sTat-ND2 310-321 was administered intravenously via the tail vein at 1 nmol / g intravenously after 8-15 days of surgical implantation of the neural cuff. After the animals were injected, they were returned to us.

Measurement of Foot Avoidance Threshold : Pitcher et al. (Journal of Neuroscience Methods, 1999), the animals were placed on a platform containing 1.5 mm diameter holes in a clear plexiglass observation chamber (30 x 30 x 30 cm). A series of elevated von Frey filaments (Stoelting, USA) (. 008g-15g) was applied to the plantar surface of the hind paw to determine the minimum stimulus required to induce foot evasion. Each filament was applied vertically for 2 seconds or until evasion occurred. Each successive plenum was applied five times at 5 second intervals. Thresholds were determined by the filament that caused 3 positive reactions during 5 applications. No filaments larger than 15g were used to avoid tissue damage. Foot avoidance thresholds were measured before neural contraction, 7 days after sciatic nerve contraction, and 45, 90, 135 and 180 minutes after injection of peptide or control.

Example  # 8: Effective to reduce pain ND2  Inhibitor

Peripheral nerve injury (PNI) via a chronic sciatic nerve shrinkage model was induced in rodents by surgically implanting a polyethylene cuff (2 mm length) around the sciatic nerve of the rat or mouse under isoflurane anesthesia. Afterwards, the inventors used von-Frey filaments as pre-operative and post-operative 8-14 days for baseline as previously described (Liu et al., 2008) The threshold value (PWT) was measured. We prepared myr-ND2 (100 micromol / L) in 20% acetic acid as a stock solution and diluted the stock to the working concentration before testing. We injected 10 pmol or 100 pmol of myr-ND2 and vehicle into the spinal cord and underwent PWT testing at 45, 90, 135 and 180 min after injection. Both concentrations of myr-ND2 tested showed reduced sensitivity to pain over all time points tested (Figure 23). Thus, ND2 peptides comprising myr-ND2 are effective inhibitors of pain.

Example  # 9: maintain efficacy in pain and stroke models without decreasing blood pressure at high dose levels Mystoise

ND2 peptides have been found herein to be effective in reducing both pain and injury after stroke. Tat peptides have previously been shown to reduce blood pressure when administered rapidly at high doses. The present inventors have tested the effect of applying rapid bolus of myr-ND2 peptides at higher concentrations than Tat-ND2 and NA-1 (Tat-NR2B9c). Rats with surgically implanted devices to measure arterial pressure were administered ~25 mg / kg of each peptide as bolus injections into the tail vein after 10 minutes of monitoring. Both peptides containing the Tat sequence reduced the mean arterial blood pressure to about half the normal level. This pressure drop was resolved within about 10-15 minutes without intervention. However, the myr-ND2 peptide did not show a decrease in blood pressure at similar concentrations in any of the animals tested (Figure 20). Figure 21 shows the lowest point in blood pressure, indicating that myr-ND2 is not effective against blood pressure in the model. Thus, myr-ND2 can be administered at a higher dose level than Tat-ND2 in humans with low blood pressure side effects or low potential for adverse blood pressure observations.

references:

Although the present invention has been described in detail for purposes of clarity of understanding, certain modifications may be practiced within the scope of the appended claims. All publications cited in this application (e.g., journal articles, registration numbers, websites, etc.) and patent literature are incorporated herein by reference in their entirety for all purposes to the same extent as if each were individually indicated. Where different sequences can be associated with the same registration number at different time points, this means the sequence associated with the registration number at the effective filing date. Effective filing date means the earliest priority date at which a pending registration number begins. Any element, embodiment, step, feature or aspect of the present invention may be performed in combination with any other element, embodiment, step, feature or aspect, unless the context clearly indicates otherwise.

Sequence List

                               SEQUENCE LISTING <110> NONO, INC.   <120> ND2 PEPTIDES AND METHODS OF TREATING NEUROLOGICAL DISEASE <130> 057769-410646 <140> PCT / US2011 / 053764 <141> 2011-09-28 <150> 61 / 387,439 <151> 2010-09-28 <160> 62 <170> PatentIn version 3.5 <210> 1 <211> 10 <212> PRT <213> Human immunodeficiency virus <400> 1 Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg 1 5 10 <210> 2 <211> 11 <212> PRT <213> Human immunodeficiency virus <400> 2 Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg 1 5 10 <210> 3 <211> 11 <212> PRT <213> Human immunodeficiency virus <400> 3 Phe Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg 1 5 10 <210> 4 <211> 12 <212> PRT <213> Human immunodeficiency virus <400> 4 Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro Gln 1 5 10 <210> 5 <211> 10 <212> PRT <213> Homo sapiens <400> 5 Lys Pro Ala Ser Ala Asp Gly His Arg Gly 1 5 10 <210> 6 <211> 10 <212> PRT <213> Homo sapiens <400> 6 Gly Ala Ala Lys Arg Pro Ser Asp Gly His 1 5 10 <210> 7 <211> 19 <212> PRT <213> Homo sapiens <400> 7 Lys Pro Ala Ser Ala Asp Gly His Arg Gly Pro Ser Ala Ala Phe Val 1 5 10 15 Pro Ala Ala              <210> 8 <211> 19 <212> PRT <213> Homo sapiens <400> 8 Ala Gly Ser His Ala Pro Phe Pro Ser Pro Ala Arg Ala Gly Val Ala 1 5 10 15 Pro Asp Ala              <210> 9 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic       peptide <400> 9 Lys Pro Ala Ser Ala Asp Gly His Arg Gly Tyr Gly Arg Lys Lys Arg 1 5 10 15 Arg Gln Arg Arg Arg             20 <210> 10 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic       peptide <400> 10 Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Lys Pro Ala Ser Ala 1 5 10 15 Asp Gly His Arg Gly             20 <210> 11 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic       peptide <400> 11 Gly Ala Ala Lys Arg Pro Ser Gly Asp His Tyr Gly Arg Lys Lys Arg 1 5 10 15 Arg Gln Arg Arg Arg             20 <210> 12 <211> 10 <212> PRT <213> Homo sapiens <400> 12 Gly Ala Phe Pro Ala Ser Gln Thr Pro Ser 1 5 10 <210> 13 <211> 20 <212> PRT <213> Homo sapiens <400> 13 Gly Ala Phe Pro Ala Ser Gln Thr Pro Ser Lys Pro Ala Ser Ala Asp 1 5 10 15 Gly His Arg Gly             20 <210> 14 <211> 15 <212> PRT <213> Homo sapiens <400> 14 Ser Gln Thr Pro Ser Lys Pro Ala Ser Ala Asp Gly His Arg Gly 1 5 10 15 <210> 15 <211> 14 <212> PRT <213> Homo sapiens <400> 15 Pro Ala Ser Ala Asp Gly His Arg Gly Pro Ser Ala Ala Phe 1 5 10 <210> 16 <211> 33 <212> PRT <213> Homo sapiens <400> 16 Asn Leu Tyr Phe Tyr Leu Arg Leu Ile Tyr Ser Thr Ser Ile Thr Leu 1 5 10 15 Leu Pro Met Ser Asn Asn Val Lys Met Lys Trp Gln Phe Glu His Thr             20 25 30 Lys      <210> 17 <211> 21 <212> PRT <213> Homo sapiens <400> 17 Asn Leu Tyr Phe Tyr Leu Arg Leu Ile Tyr Ser Thr Ser Ile Thr Leu 1 5 10 15 Leu Pro Met Ser Asn             20 <210> 18 <211> 13 <212> PRT <213> Homo sapiens <400> 18 Tyr Phe Tyr Leu Arg Leu Ile Tyr Ser Thr Ser Ile Thr 1 5 10 <210> 19 <211> 24 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic       peptide <400> 19 Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Tyr Phe Tyr Leu Arg 1 5 10 15 Leu Ile Tyr Ser Thr Ser Ile Thr             20 <210> 20 <211> 20 <212> PRT <213> Homo sapiens <400> 20 Ser Thr Ser Ile Thr Leu Leu Pro Met Ser Asn Asn Val Lys Met Lys 1 5 10 15 Trp Gln Phe Glu             20 <210> 21 <211> 20 <212> PRT <213> Homo sapiens <400> 21 Ile Thr Leu Leu Pro Met Ser Asn Asn Val Lys Met Lys Trp Gln Phe 1 5 10 15 Glu His Thr Lys             20 <210> 22 <211> 15 <212> PRT <213> Homo sapiens <400> 22 Met Ser Asn Asn Val Lys Met Lys Trp Gln Phe Glu His Thr Lys 1 5 10 15 <210> 23 <211> 12 <212> PRT <213> Homo sapiens <400> 23 Asn Val Lys Met Lys Trp Gln Phe Glu His Thr Lys 1 5 10 <210> 24 <211> 12 <212> PRT <213> Homo sapiens <400> 24 Lys Trp Val Gln His Thr Lys Phe Glu Met Lys Asn 1 5 10 <210> 25 <211> 8 <212> PRT <213> Homo sapiens <400> 25 Lys Trp Gln Phe Glu His Thr Lys 1 5 <210> 26 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic       peptide <400> 26 Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Asn Val Lys Met Lys 1 5 10 15 Trp Gln Phe Glu His Thr Lys             20 <210> 27 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic       peptide <400> 27 Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Lys Trp Val Gln His 1 5 10 15 Thr Lys Phe Glu Met Lys Asn             20 <210> 28 <211> 12 <212> PRT <213> Homo sapiens <400> 28 Met Ser Asn Asn Val Lys Met Lys Trp Gln Phe Glu 1 5 10 <210> 29 <211> 9 <212> PRT <213> Homo sapiens <400> 29 Asn Val Lys Met Lys Trp Gln Phe Glu 1 5 <210> 30 <211> 7 <212> PRT <213> Homo sapiens <400> 30 Asn Val Lys Met Lys Trp Gln 1 5 <210> 31 <211> 5 <212> PRT <213> Homo sapiens <400> 31 Asn Val Lys Met Lys 1 5 <210> 32 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic       peptide <400> 32 Asn Val Lys Met Lys Trp Gln Phe Glu His Thr Lys 1 5 10 <210> 33 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic       peptide <400> 33 Met Ser Asn Asn Val Lys Met Lys Trp Gln Phe Glu His Thr Lys 1 5 10 15 <210> 34 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic       peptide <220> <221> MOD_RES <222> (1) <223> Any amino acid or not present <400> 34 Xaa Gly Lys Lys Lys Lys Lys Lys Gln Lys Lys Lys 1 5 10 <210> 35 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic       peptide <220> <221> MOD_RES <222> (1) <223> Any amino acid or not present <400> 35 Xaa Arg Lys Lys Arg Arg Gln Arg Arg Arg 1 5 10 <210> 36 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic       peptide <220> <221> MOD_RES <222> (1) <223> Any amino acid or not present <400> 36 Xaa Gly Ala Lys Lys Arg Arg Gln Arg Arg Arg 1 5 10 <210> 37 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic       peptide <220> <221> MOD_RES <222> (1) <223> Any amino acid or not present <400> 37 Xaa Ala Lys Lys Arg Arg Gln Arg Arg Arg 1 5 10 <210> 38 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic       peptide <220> <221> MOD_RES <222> (1) <223> Any amino acid or not present <400> 38 Xaa Gly Arg Lys Ala Arg Arg Gln Arg Arg Arg 1 5 10 <210> 39 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic       peptide <220> <221> MOD_RES <222> (1) <223> Any amino acid or not present <400> 39 Xaa Arg Lys Ala Arg Arg Gln Arg Arg Arg 1 5 10 <210> 40 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic       peptide <220> <221> MOD_RES <222> (1) <223> Any amino acid or not present <400> 40 Xaa Gly Arg Lys Lys Ala Arg Gln Arg Arg Arg 1 5 10 <210> 41 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic       peptide <220> <221> MOD_RES <222> (1) <223> Any amino acid or not present <400> 41 Xaa Arg Lys Lys Ala Arg Gln Arg Arg Arg 1 5 10 <210> 42 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic       peptide <220> <221> MOD_RES <222> (1) <223> Any amino acid or not present <400> 42 Xaa Gly Arg Lys Lys Arg Arg Gln Ala Arg Arg 1 5 10 <210> 43 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic       peptide <220> <221> MOD_RES <222> (1) <223> Any amino acid or not present <400> 43 Xaa Arg Lys Lys Arg Arg Gln Ala Arg Arg 1 5 10 <210> 44 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic       peptide <400> 44 Gly Ser Ser Ser Ser 1 5 <210> 45 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic       peptide <400> 45 Thr Gly Glu Lys Pro 1 5 <210> 46 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic       peptide <400> 46 Gly Gly Arg Arg Gly Gly Gly Ser 1 5 <210> 47 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic       peptide <400> 47 Leu Arg Gln Arg Asp Gly Glu Arg Pro 1 5 <210> 48 <211> 13 <212> PRT <213> Human immunodeficiency virus <400> 48 Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro Gln 1 5 10 <210> 49 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic       peptide <220> <221> MOD_RES <222> (1) <223> Any amino acid other than "Y" or not present <400> 49 Xaa Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg 1 5 10 <210> 50 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic       peptide <220> <221> MOD_RES <222> (1) <223> Any amino acid or not present <400> 50 Xaa Gly Arg Lys Lys Arg Arg Gln Arg Ala Arg 1 5 10 <210> 51 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic       peptide <220> <221> MOD_RES <222> (1) <223> Any amino acid or not present <400> 51 Xaa Arg Lys Lys Arg Arg Gln Arg Ala Arg 1 5 10 <210> 52 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic       peptide <220> <221> MOD_RES <222> (1) <223> Any amino acid or not present <400> 52 Xaa Arg Arg Pro Arg Arg Pro Arg Arg Pro Arg Arg 1 5 10 <210> 53 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic       peptide <220> <221> MOD_RES <222> (1) <223> Any amino acid or not present <400> 53 Xaa Arg Arg Ala Arg Arg Ala Arg Arg Ala Arg Arg 1 5 10 <210> 54 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic       peptide <220> <221> MOD_RES <222> (1) <223> Any amino acid or not present <400> 54 Xaa Arg Arg Arg Ala Arg Arg Arg Ala Arg Arg 1 5 10 <210> 55 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic       peptide <220> <221> MOD_RES <222> (1) <223> Any amino acid or not present <400> 55 Xaa Arg Arg Arg Arg Pro Arg Arg Arg Arg Arg Arg 1 5 10 <210> 56 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic       peptide <220> <221> MOD_RES <222> (1) <223> Any amino acid or not present <400> 56 Xaa Arg Arg Pro Arg Arg Pro Arg Arg 1 5 <210> 57 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic       peptide <220> <221> MOD_RES <222> (1) <223> Any amino acid or not present <400> 57 Xaa Arg Arg Ala Arg Arg Ala Arg Arg 1 5 <210> 58 <211> 11 <212> PRT <213> Human immunodeficiency virus <400> 58 Arg Arg Arg Gln Arg Arg Lys Lys Arg Gly Tyr 1 5 10 <210> 59 <211> 11 <212> PRT <213> Human immunodeficiency virus <400> 59 Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro 1 5 10 <210> 60 <211> 347 <212> PRT <213> Homo sapiens <400> 60 Met Asn Pro Leu Ala Gln Pro Val Ile Tyr Ser Thr Ile Phe Ala Gly 1 5 10 15 Thr Leu Ile Thr Ala Leu Ser Ser His Trp Phe Phe Thr Trp Val Gly             20 25 30 Leu Glu Met Asn Met Leu Ala Phe Ile Pro Val Leu Thr Lys Lys Met         35 40 45 Asn Pro Arg Ser Thr Glu Ala Ala Ile Lys Tyr Phe Leu Thr Gln Ala     50 55 60 Thr Ala Ser Met Ile Leu Leu Met Ala Ile Leu Phe Asn Asn Met Leu 65 70 75 80 Ser Gly Gln Trp Thr Met Thr Asn Thr Thr Asn Gln Tyr Ser Ser Leu                 85 90 95 Met Ile Met Met Ala Met Ala Met Lys Leu Gly Met Ala Pro Phe His             100 105 110 Phe Trp Val Pro Glu Val Thr Gln Gly Thr Pro Leu Thr Ser Gly Leu         115 120 125 Leu Leu Leu Thr Trp Gln Lys Leu Ala Pro Ile Ser Ile Met Tyr Gln     130 135 140 Ile Ser Pro Ser Leu Asn Val Ser Leu Leu Leu Thr Leu Ser Ile Leu 145 150 155 160 Ser Ile Met Ala Gly Ser Trp Gly Gly Leu Asn Gln Thr Gln Leu Arg                 165 170 175 Lys Ile Leu Ala Tyr Ser Ser Ile Thr His Met Gly Trp Met Met Ala             180 185 190 Val Leu Pro Tyr Asn Pro Asn Met Thr Ile Leu Asn Leu Thr Ile Tyr         195 200 205 Ile Ile Leu Thr Thr Thr Ala Phe Leu Leu Leu Asn Leu Asn Ser Ser     210 215 220 Thr Thr Leu Leu Leu Ser Arg Thr Trp Asn Lys Leu Thr Trp Leu 225 230 235 240 Thr Pro Leu Ile Pro Ser Thr Leu Leu Ser Leu Gly Gly Leu Pro Pro                 245 250 255 Leu Thr Gly Phe Leu Pro Lys Trp Ala Ile Ile Glu Glu Phe Thr Lys             260 265 270 Asn Asn Ser Leu Ile Ile Pro Thr Ile Met Ala Thr Ile Thr Leu Leu         275 280 285 Asn Leu Tyr Phe Tyr Leu Arg Leu Ile Tyr Ser Thr Ser Ile Thr Leu     290 295 300 Leu Pro Met Ser Asn Asn Val Lys Met Lys Trp Gln Phe Glu His Thr 305 310 315 320 Lys Pro Thr Pro Phe Leu Pro Thr Leu Ile Ala Leu Thr Thr Leu Leu                 325 330 335 Leu Pro Ile Ser Pro Phe Met Leu Met Ile Leu             340 345 <210> 61 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic       peptide <220> <221> MOD_RES <222> (1) <223> Any amino acid or not present <400> 61 Xaa Phe Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg 1 5 10 <210> 62 <211> 20 <212> PRT <213> Human immunodeficiency virus <400> 62 Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Lys Leu Ser Ser Ile 1 5 10 15 Glu Ser Asp Val             20

Claims (37)

An ND2 peptide consisting of 20 or fewer residues identical to the residue of SEQ ID NO: 60, containing residues 310 to 321 of SEQ ID NO: 60, wherein said peptide inhibits ND2 interaction with Src. The method of claim 1, wherein the ND2 peptide is an ND2 peptide, wherein residues 310 to 321 of SEQ ID NO: 60 or residues 307 to 321 of SEQ ID NO: 60 3. The ND2 peptide of claim 2, wherein the ND2 peptide is myristoylated at the N-terminus. 2. The ND2 peptide of claim 1, wherein the ND2 peptide is lipidated. 3. The ND2 peptide of claim 1, wherein the ND2 peptide is linked to an internalizing peptide. The ND2 peptide of claim 5 linked to an internalizing peptide as a chimeric peptide, wherein the chimeric peptide is 50 amino acids or less in length. 7. The ND2 peptide of claim 6, wherein the chimeric peptide is less than or equal to 25 amino acids in length. A pharmaceutical composition for use in treating or preventing a stroke, comprising the chimeric peptide or the ND2 peptide of any one of claims 1 to 7. A pharmaceutical composition for use in treating or preventing pain, comprising the chimeric peptide or the ND2 peptide of any one of claims 1 to 7. delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete

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